Alpha polyglutamated antifolates and uses thereof

ABSTRACT

The disclosure relates generally to alpha polyglutamated Antifolates, formulations containing liposomes filled with alpha polyglutamated Antifolates, methods of making the alpha polyglutamated Antifolates and liposome containing formulations, and methods of using polyglutamated alpha polyglutamated Antifolates and liposome containing formulations to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., an autoimmune disease such as rheumatoid arthritis).

BACKGROUND

This disclosure generally relates to alpha polyglutamated Antifolatecompositions, including delivery vehicles such as liposomes containingthe alpha polyglutamated Antifolate compositions, and methods of makingand using the compositions to treat diseases includinghyperproliferative diseases such as cancer, disorders of the immunesystem including inflammation and autoimmune diseases such as rheumatoidarthritis, and infectious diseases such as HIV, malaria, andschistomiasis.

Folate is an essential cofactor that mediates the transfer of one-carbonunits involved in nucleotide biosynthesis and DNA repair, theremethylation of homocysteine (Hcy), and the methylation of DNA,proteins, and lipids. The only circulating forms of folates in the bloodare monoglutamates and folate monoglutamates are the only form of folatethat is transported across the cell membrane—likewise, the monoglutamateform of polyglutamatable antifolates is transported across the cellmembrane. Once taken up into cells, intracellular folate is converted topolyglutamates by the enzyme folylpoly-gamma-glutamate synthetase(FPGS).

Antifolate is transported into cells by the reduced folate carrier (RFC)system and folate receptors (FRs) α and β and by Proton Coupled FolateTransporter (PCFT) that is generally most active in a lower pHenvironment. RFC is the main transporter of antifolate at physiologic pHand is ubiquitously expressed in both normal and diseased cells.Consequently, antifolate treatment often suffers from the dose-limitingtoxicity that is a major obstacle in cancer chemotherapy. Once insidethe cell, antifolate is polyglutamated by FPGS, which may add up to 6glutamyl groups in an L-gamma carboxyl group linkage to the antifolate.The L-gamma polyglutamation of antifolates by FPGS serves at least 2main therapeutic purposes: (1) it greatly enhances antifolate affinityand inhibitory activity for DHFR; and (2) it facilitates theaccumulation of polyglutamated antifolate, which unlike antifolate(monoglutamate), is not easily transported out of cells by cell effluxpumps.

While targeting folate metabolism and nucleotide biosynthesis is awell-established therapeutic strategy for cancer, for antifolate,clinical efficacy is limited by a lack of tumor selectivity and thepresence of de novo and acquired drug resistance. Antifolates often actduring DNA and RNA synthesis, and consequently have a greater toxiceffect on rapidly dividing cells such as malignant and myeloid cells.Myelosuppression is typically the dose-limiting toxicity of antifolatetherapy and has limited the clinical applications of antifolates.

Resistance to antifolate therapy is typically associated with one ormore of, (a) increased cell efflux pump activity, (b) decreasedtransport of antifolates into cells (c) increased DHFR activity, (d)decreased folylpoly-gamma-glutamate synthetase (FPGS) activity, and (e)increased gamma-glutamyl hydrolase (GGH) activity, which cleaves gammapolyglutamate chains attached to folates and antifolates.

The challenge to the longstanding (>30 years) observation thathigher-level polyglutamates of various antifolates have much greaterpotency compared to lower-level glutamates, has been that the scientificcommunity has relied on the intracellular FPGS mediated mechanisms toconvert the lower-level glutamates to their higher-level forms. Thepresent inventions provide the means to deliver higher-levelpolyglutamate forms of antifolates directly into the cell, withouthaving to rely on the cells' machinery to achieve this goal.

The provided alpha polyglutamated Antifolate compositions deliver astrategy for overcoming the pharmacological challenges associated withthe dose limiting toxicities and with treatment resistance associatedwith Antifolate therapy. In some embodiments, the provided methodsdeliver to cancer cells an alpha polyglutamated form of an Antifolatewhile (1) minimizing/reducing exposure to normal tissue cells, (2)optimizing/improving the cytotoxic effect of antifolate-based agents oncancer cells and (3) minimizing/reducing the impact of the efflux pumps,and other resistance mechanisms that limit the therapeutic efficacy ofantifolates.

BRIEF SUMMARY

This disclosure generally relates to novel alpha polyglutamatedAntifolate (αPANTIFOL) compositions and methods of making and using thecompositions to treat diseases including hyperproliferative diseasessuch as cancer, disorders of the immune system such as inflammation andrheumatoid arthritis, cardiovascular disease such as coronary arterydisease, and infectious disease such as HIV, malaria, and schistomiasis.

In some embodiments, the disclosure provides:

-   [1] a composition comprising an alpha polyglutamated Antifolate,    wherein at least one glutamyl group has an alpha carboxyl group    linkage;-   [2] the composition of [1], wherein the Antifolate is selected from:    piritrexim, pralatrexate, AG2034, GW1843, and LY309887, or a    stereoisomer thereof;-   [3] the composition of [1], wherein the Antifolate is selected from:    PMX, MTX, RTX, and LMX, or a stereoisomer thereof;-   [4] the composition according to [1], wherein the Antifolate is    selected from: LV (etoposide), L-leucovorin    (L-5-formyltetrahydrofolate); 5-CH3-THF, 5-methyltetrahydrofolate;    FA, folic acid; PteGlu, pteroyl glutamate (FA); MTX, methotrexate;    2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT,    Antifolate; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT,    2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin;    PT523, N alpha-(4-amino-4-deoxypteroyl)-N    delta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),    5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,    5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,    N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, N    alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N    alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid;    5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;    5-dH4PteAPBA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phospho-butanoic    acid; 5-dH4PteOro, N    alpha-(5-deaza-5,6,7,8-tetrahydropt-eroyl)-L-ornithine; CB3717,    N10-propargyl-5,8-dideazafolic acid; ICI-198,583,    2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;    4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,    4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;    Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583;    7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694,    N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic    acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,    (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaric    acid; LY231514,    N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic    acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;    2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu,    5-deazaaisofolic acid; N9-CH3-5-d(i)PteGlu,    N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu,    N9-formyl-5-deazaisofolic acid; AG337,    3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline;    and 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)    amino]quinazoline; or a stereoisomer thereof;-   [5] the composition of [1], wherein the Antifolate is selected from:    methotrexate, raltitrexed, plevitrexed, pemetrexed, lometrexol (LMX;    5,10-dideazatetrahydrofolic acid), a cyclopenta[g]quinazoline with a    dipeptide ligand, CB3717, CB300945, or a stereoisomer thereof, such    as 6-R,S-BGC 945 (ONX-0801), CB300638, and BW1843U89;-   [6] the composition according to any of [1]-[5], wherein,    -   (a) each of the glutamyl groups of the polyglutamated Antifolate        other than the glutamyl group of the Antifolate has an alpha        carboxyl group linkage; or    -   (b) two or more glutamyl groups of the polyglutamated Antifolate        have a gamma carboxyl group linkage;-   [7] the composition according to any of [1]-[5], wherein,    -   (a) each of the glutamyl groups other than the C-terminal        glutamyl group or groups and the glutamyl group of the        Antifolate has an alpha carboxyl group linkage; or    -   (b) each of the glutamyl groups other than the C-terminal        glutamyl group or groups has an alpha carboxyl group linkage;-   [8] the composition according to any of [1]-[7], wherein the alpha    polyglutamated Antifolate:    -   (a) contains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl        groups;    -   (b) is an alpha pentaglutamated Antifolate; or    -   (c) is an alpha hexaglutamated Antifolate;-   [9] the composition according to any of [1]-[8], wherein the alpha    polyglutamated Antifolate comprises 1-10 glutamyl groups having an    alpha carboxyl group linkage;-   [10] the composition according to any of [1]-[9], wherein:    -   (a) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form,    -   (b) each of the glutamyl groups of the alpha polyglutamated        Antifolate is in the L-form,    -   (c) at least 1 of the glutamyl groups of the alpha        polyglutamated Antifolate is in the D-form,    -   (d) each of the glutamyl groups of the alpha polyglutamated        Antifolate other than the glutamyl group of the Antifolate is in        the D-form, or    -   (e) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form and at least 1 of        the glutamyl groups is in the D-form;-   [11] the composition according to any of [1]-[10], wherein the    polyglutamate is linear;-   [12] the composition according to any of [1]-[10], wherein the    polyglutamate is branched;-   [13] a liposomal composition comprising the alpha polyglutamated    Antifolate according to any of [1]-[12] (Lp-αPANTIFOL);-   [14] the Lp-αPANTIFOL composition of [13], wherein the alpha    polyglutamated Antifolate is selected from:    -   (a) AG2034, piritrexim, pralatrexate, GW1843, Antifolate, and        LY309887; or    -   (b) PMX, MTX, RTX, and LMX, or a stereoisomer thereof;-   [15] the Lp-αPANTIFOL composition of [13], wherein the    polyglutamated Antifolate is selected from: LV (etoposide),    L-leucovorin (L-5-formyltetrahydrofolate); 5-CH3-THF,    5-methyltetrahydrofolate; FA, folic acid; PteGlu, pteroyl glutamate    (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX,    2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT;    2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,    10-ethyl-10-deazaaminopterin; PT523, N    alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine;    DDATHF (lometrexol), 5,10-dideaza-5,6,7,8-tetrahydrofolic acid;    5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroisofolic acid;    N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic    acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid;    5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic    acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine;    5-dH4PteHCysA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;    5-dH4PteAPBA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoic    acid; 5-dH4PteOro, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,    N10-propargyl-5,8-dideazafolic acid; ICI-198,583,    2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;    4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,    4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;    Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583;    7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694,    N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic    acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,    (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaric    acid; LY231514,    N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic    acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;    2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu,    5-deazaaisofolic acid; N9-CH3-5-d(i)PteGlu,    N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu,    N9-formyl-5-deazaisofolic acid; AG337,    3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline;    and AG377,    2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline;    or a stereoisomer thereof;-   [16] the Lp-αPANTIFOL composition according to [13], wherein the    Antifolate is selected from: methotrexate, raltitrexed, plevitrexed,    pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), a    cyclopenta[g]quinazoline with a dipeptide ligand, CB3717, CB300945,    or a stereoisomer thereof, such as 6-R,S-BGC 945 (ONX-0801),    CB300638, and BW1843U89;-   [17] the Lp-αPANTIFOL composition according to any of [13]-[16],    wherein the liposome comprises an alpha polyglutamated Antifolate    containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups;-   [18] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha tetraglutamated Antifolate;-   [19] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha pentaglutamated Antifolate;-   [20] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha hexaglutamated Antifolate;-   [21] the Lp-αPANTIFOL composition according to any of [13]-[20],    wherein the polyglutamate is linear or branched;-   [22] the Lp-αPANTIFOL composition according to any of [13]-[21],    wherein:    -   (a) each of the glutamyl groups other than the glutamyl group of        the Antifolate has an alpha carboxyl group linkage, or    -   (b) two or more glutamyl groups have a gamma carboxyl group        linkage;-   [23] the Lp-αPANTIFOL composition according to any of [13]-[21],    wherein:    -   (a) each of the glutamyl groups other than the C-terminal        glutamyl group or groups and the glutamyl group of the        Antifolate has an alpha carboxyl group linkage, or    -   (b) each of the glutamyl groups other than the C-terminal        glutamyl group or groups has an alpha carboxyl group linkage;-   [24] the Lp-αPANTIFOL composition according to any of [13]-[23],    wherein    -   (a) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form,    -   (b) each of the glutamyl groups of the alpha polyglutamated        Antifolate is in the L-form,    -   (c) at least 1 of the glutamyl groups of the alpha        polyglutamated Antifolate is in the D-form,    -   (d) each of the glutamyl groups of the alpha polyglutamated        Antifolate other than the glutamyl group of the Antifolate is in        the D-form, or    -   (e) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form and at least 1 of        the glutamyl groups is in the D-form;-   [25] the Lp-αPANTIFOL composition according to any of [13]-[24],    wherein the liposome is pegylated (PαLp-αPANTIFOL);-   [26] the Lp-αPANTIFOL composition according to any of [13]-[24],    wherein the liposome is not pegylated;-   [27] the Lp-αPANTIFOL composition according to any of [13]-[26],    wherein the liposome has a diameter in the range of 20 nm to 200 nm;-   [28] the Lp-αPANTIFOL composition according to any of [13]-[27],    wherein the polyglutamate is linear or branched;-   [29] the Lp-αPANTIFOL composition according to any of [13]-[28],    wherein the liposomes comprise at least 1% weight by weight (w/w) of    the alpha polyglutamated Antifolate or wherein during the process of    preparing the Lp-αPANTIFOL, at least 1% of the starting material of    alpha polyglutamated Antifolate is encapsulated (entrapped) in the    Lp-αPANTIFOL;-   [30] the Lp-αPANTIFOL composition according to any of [13]-[29],    wherein the liposome has a diameter in the range of 20 nm to 500 nm    or 20 nm to 200 nm;-   [31] the Lp-αPANTIFOL composition according to any of [13]-[29],    wherein the liposome has a diameter in the range of 80 nm to 120 nm;-   [32] the Lp-αPANTIFOL composition according to any of [13]-[31],    wherein the liposome is formed from liposomal components;-   [33] the Lp-αPANTIFOL composition according to [32], wherein the    liposomal components comprise at least one of an anionic lipid and a    neutral lipid;-   [34] the Lp-αPANTIFOL composition according to [32] or [33], wherein    the liposomal components comprise at least one selected from: DSPE;    DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol;    cholesterol-PEG; and cholesterol-maleimide;-   [35] the Lp-αPANTIFOL composition according to any of [32]-[34],    wherein the liposomal components comprise at least one selected    from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide;    cholesterol; and HSPC;-   [36] the Lp-αPANTIFOL composition according to any of [32]-[35],    wherein one or more liposomal components further comprises a steric    stabilizer;-   [37] the Lp-αPANTIFOL composition according to [36], wherein the    steric stabilizer is at least one selected from polyethylene glycol    (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl    pyrrolidone) (PVP); poly(acrylamide) (PAA);    poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl    polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic    poly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol,    copolymer containing polyethylene glycol and polypropylene oxide,    Poloxamer 188, and polyvinyl alcohol;-   [38] the Lp-αPANTIFOL composition according to [37], wherein the    steric stabilizer is PEG and the PEG has a number average molecular    weight (Mn) of 200 to 5000 daltons;-   [39] the Lp-αPANTIFOL composition according to any of [13]-[38],    wherein the liposome is anionic or neutral;-   [40] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is less than or equal    to zero;-   [41] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is between 0 to −150    mV;-   [42] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is between −30 to −50    mV;-   [43] the Lp-αPANTIFOL composition according to any of [13]-[38],    wherein the liposome is cationic;-   [44] the Lp-αPANTIFOL composition according to any of [13]-[43],    wherein the liposome has an interior space comprising the alpha    polyglutamated Antifolate and an aqueous pharmaceutically acceptable    carrier;-   [45] the Lp-αPANTIFOL composition of [44], wherein the    pharmaceutically acceptable carrier comprises a tonicity agent such    as dextrose, mannitol, glycerine, potassium chloride, sodium    chloride, at a concentration of greater than 1%;-   [46] the Lp-αPANTIFOL composition of [44], wherein the aqueous    pharmaceutically acceptable carrier is trehalose;-   [47] the Lp-αPANTIFOL composition of [46], wherein the    pharmaceutically acceptable carrier comprises 5% to 20% weight of    trehalose;-   [48] the Lp-αPANTIFOL composition according to any of [44]-[47],    wherein the pharmaceutically acceptable carrier comprises 1% to 15    weight of dextrose;-   [49] the Lp-αPANTIFOL composition according to any of [44]-[48],    wherein the interior space of the liposome comprises 5% dextrose    suspended in an HEPES buffered solution;-   [50] the Lp-αPANTIFOL composition according to any of [44]-[49],    wherein the pharmaceutically acceptable carrier comprises a buffer    such as HEPES Buffered Saline (HBS) or similar, at a concentration    of between 1 to 200 mM and a pH of between 2 to 8;-   [51] the Lp-αPANTIFOL composition according to any of [44]-[50],    wherein the pharmaceutically acceptable carrier comprises a total    concentration of sodium acetate and calcium acetate of between 50 mM    to 500 mM;-   [52] the Lp-αPANTIFOL composition according to any of [13]-[51],    wherein the interior space of the liposome has a pH of 5-8 or a pH    of 6-7, or any range therein between;-   [53] the Lp-αPANTIFOL composition according to any of [13]-[52],    wherein the liposome comprises less than 500,000 or less than    200,000 molecules of the alpha polyglutamated Antifolate;-   [54] the Lp-αPANTIFOL composition according to any of [13]-[53],    wherein the liposome comprises between 10 to 100,000 molecules of    the alpha polyglutamated Antifolate, or any range therein between;-   [55] the Lp-αPANTIFOL composition according to any of [13]-[54],    which further comprises a targeting moiety and wherein the targeting    moiety has a specific affinity for a surface antigen on a target    cell of interest;-   [56] the Lp-αPANTIFOL composition according to [55], wherein the    targeting moiety is attached to one or both of a PEG and the    exterior of the liposome, optionally wherein targeting moiety is    attached to one or both of the PEG and the exterior of the liposome    by a covalent bond;-   [57] the Lp-αPANTIFOL composition of [55] or [56], wherein the    targeting moiety is a polypeptide;-   [58] the Lp-αPANTIFOL composition according to any of [55]-[57],    wherein the targeting moiety is an antibody or an antigen binding    fragment of an antibody;-   [59] the Lp-αPANTIFOL composition according to any of [55]-[58],    wherein the targeting moiety binds the surface antigen with an    equilibrium dissociation constant (Kd) in a range of 0; 5×10-10 to    10×10-6 as determined using BIACORE® analysis;-   [60] the Lp-αPANTIFOL composition according to any of [55]-[59],    wherein the targeting moiety specifically binds one or more folate    receptors selected from: folate receptor alpha (FR-α), folate    receptor beta (FR-β), and folate receptor delta (FR-δ);-   [61] the Lp-αPANTIFOL composition according to any of [55]-[60],    wherein the targeting moiety comprises one or more selected from: an    antibody, a humanized antibody, an antigen binding fragment of an    antibody, a single chain antibody, a single-domain antibody, a    bi-specific antibody, a synthetic antibody, a pegylated antibody,    and a multimeric antibody;-   [62] the Lp-αPANTIFOL composition according to any of [55]-[61],    wherein each pegylated liposome comprises from 1 to 1000 or 30-200    targeting moieties;-   [63] the Lp-αPANTIFOL composition according to any of [44]-[57],    further comprising one or more of an immunostimulatory agent, a    detectable marker and a maleimide, wherein the immunostimulatory    agent, the detectable marker or the maleimide is attached to said    PEG or the exterior of the liposome;-   [64] the Lp-αPANTIFOL composition of [63], wherein the    immunostimulating agent is at least one selected from: a protein    immunostimulating agent; a nucleic acid immunostimulating agent; a    chemical immunostimulating agent; a hapten; and an adjuvant;-   [65] the Lp-αPANTIFOL composition of [63] or [64], wherein the    immunostimulating agent is at least one selected from: a    fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta    glucan; a beta-1,3-glucan; a beta-1,6-glucan; a resolvin (e.g., a    Resolvin D such as Dn-6DPA or Dn-3DPA, a Resolvin E. or a T series    resolvin); and a Toll-like receptor (TLR) modulating agent such as,    an oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and an    eritoran lipid (e.g., E5564);-   [66] the Lp-αPANTIFOL composition according to any of [63]-[65],    wherein the immunostimulatory agent and the detectable marker is the    same;-   [67] the Lp-αPANTIFOL composition according to any of [63]-[66],    further comprising a hapten;-   [68] the Lp-αPANTIFOL composition of [67], wherein the hapten    comprises one or more of fluorescein or Beta 1, 6-glucan;-   [69] the Lp-αPANTIFOL composition according to any of [13]-[68],    which further comprises at least one cryoprotectant selected from    mannitol; trehalose; sorbitol; and sucrose;-   [70] a targeted composition comprising the composition according to    any of [1]-[69];-   [71] an non-targeted composition comprising the composition    according to any of [1]-[54] and [64]-[69];-   [72] the Lp-αPANTIFOL composition according to any of [13]-[71],    which further comprises carboplatin and/or pembroluzumab;-   [73] a pharmaceutical composition comprising the liposomal alpha    polyglutamated Antifolate composition according to any of [13]-[72];-   [74] a pharmaceutical composition comprising alpha polyglutamated    Antifolate composition according to any of [1]-[8];-   [75] the composition of any of [1]-[74], for use in the treatment of    disease;-   [76] use of the composition of any of [1]-[75], in the manufacture    of a medicament for the treatment of disease;-   [77] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the composition of any of [1]-[75] to the subject;-   [78] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74] to the subject;-   [79] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the composition of any of    [1]-[74];-   [80] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the liposomal alpha    polyglutamated Antifolate composition of any of [13]-[74];-   [81] the method of [79] or [80], wherein the hyperproliferative cell    is a cancer cell, a mammalian cell, and/or a human cell;-   [82] a method for treating cancer that comprises administering an    effective amount of the composition of any of [1]-[74] to a subject    having or at risk of having cancer;-   [83] a method for treating cancer that comprises administering an    effective amount of the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[73] to a subject having or at risk of    having cancer;-   [84] the method of [82] or [83], wherein the cancer is selected    from: a non-hematologic malignancy including such as for example,    lung cancer, pancreatic cancer, breast cancer, ovarian cancer,    prostate cancer, head and neck cancer, gastric cancer,    gastrointestinal cancer, colorectal cancer, esophageal cancer,    cervical cancer, liver cancer, kidney cancer, biliary duct cancer,    gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma),    brain cancer, central nervous system cancer, and melanoma; and a    hematologic malignancy such as for example, a leukemia, a lymphoma    and other B cell malignancies, myeloma and other plasma cell    dyscrasias;-   [85] the method of [82] or [83], wherein the cancer is selected    from: the cancer is a member selected from: breast cancer, advanced    head and neck cancer, lung cancer, stomach cancer, osteosarcoma,    Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL),    mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,    chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma    (desmoid tumors, aggressive fibromatosis), bladder cancer, and    Central Nervous System (CNS) lymphoma;-   [86] the method of [82] or [83], wherein the cancer is selected    from: colorectal cancer, lung cancer, breast cancer, head and neck    cancer, and pancreatic cancer;-   [87] the method of [82] or [83], wherein the cancer is a sarcoma    such as osteosarcoma;-   [88] a method for treating cancer that comprises administering an    effective amount of the Lp-αPANTIFOL composition of any of [55]-[71]    to a subject having or at risk of having a cancer cell that    expresses on its surface a folate receptor bound by the targeting    moiety;-   [89] a maintenance therapy comprising administering an effective    amount of the composition of any of [1]-[74] to a subject that is    undergoing or has undergone cancer therapy;-   [90] a maintenance therapy comprising administering an effective    amount of the liposomal alpha polyglutamated Antifolate composition    of any of [13]-[74] to a subject that is undergoing or has undergone    cancer therapy;-   [91] a method for treating a disorder of the immune system that    comprises administering an effective amount of the composition of    any of [1]-[74] to a subject having or at risk of having a disorder    of the immune system, optionally wherein the disorder of the immune    system is selected from: inflammation (e.g., acute and chronic),    systemic inflammation, rheumatoid arthritis, inflammatory bowel    disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic    lupus erythematosus, and Takayasu, and psoriasis;-   [92] a method for treating a disorder of the immune system that    comprises administering an effective amount of the liposomal alpha    polyglutamated Antifolate composition of any of [9]-[74] to a    subject having or at risk of having a disorder of the immune system,    optionally wherein the disorder of the immune system is selected    from: inflammation (e.g., acute and chronic), systemic inflammation,    rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn    disease, dermatomyositis/polymyositis, systemic lupus erythematosus,    and Takayasu, and psoriasis;-   [93] a method for treating:    -   (a) an infectious disease that comprises administering an        effective amount of the composition according to any of [1]-[74]        to a subject having or at risk of having an infectious disease;    -   (b) an infectious disease, cardiovascular disease, metabolic        disease, or another disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having an        infectious disease, cardiovascular disease, or another disease,        wherein the disease is a member selected from: atherosclerosis,        cardiovascular disease (CVD), coronary artery disease,        myocardial infarction, stroke, metabolic syndrome, a gestational        trophoblastic disease, and ectopic pregnancy;    -   (c) an autoimmune disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having an        autoimmune disease;    -   (d) rheumatoid arthritis, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having rheumatoid        arthritis;    -   (e) an inflammatory condition that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having        inflammation, optionally wherein the inflammation is acute,        chronic, and/or systemic inflammation; or    -   (f) a skin condition that comprises administering an effective        amount of the composition according to of any of claims any of        [1]-[74] to a subject having or at risk of having a skin        condition, optionally wherein the skin condition is psoriasis;-   [94] a method for treating an infectious disease that comprises    administering an effective amount of the liposomal alpha    polyglutamated Antifolate composition of any of [13]-[74] to a    subject having or at risk of having an infectious disease;-   [95] a method of delivering alpha polyglutamated Antifolate to a    tumor expressing a folate receptor on its surface, the method    comprising: administering the Lp-αPANTIFOL composition of any of    [1]-[74] to a subject having the tumor in an amount to deliver a    therapeutically effective dose of the alpha polyglutamated    Antifolate to the tumor;-   [96] a method of preparing an alpha polyglutamated Antifolate    composition comprising the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74], the method comprising: forming a    mixture comprising: liposomal components and alpha polyglutamated    antifolate in solution; homogenizing the mixture to form liposomes    in the solution; and processing the mixture to form liposomes    containing alpha polyglutamated Antifolate;-   [97] a method of preparing an alpha polyglutamated Antifolate    composition comprising the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74], the method comprising: forming a    mixture comprising: liposomal components and alpha polyglutamated    Antifolate in solution; and processing the mixture to form liposomes    containing alpha polyglutamated Antifolate;-   [98] the method of [97], wherein the processing the mixture    comprises homogenizing the mixture to form liposomes in the    solution;-   [99] a method of preparing the composition of any of [55]-[74]    comprising the steps of: forming a mixture comprising: liposomal    components and alpha polyglutamated Antifolate in a solution;    homogenizing the mixture to form liposomes in the solution;    processing the mixture to form liposomes entrapping and/or    encapsulating alpha polyglutamated Antifolate; and providing a    targeting moiety on a surface of the liposomes, the targeting moiety    having specific affinity for at least one of folate receptor alpha    (FR-α), folate receptor beta (FR-β) and folate receptor delta    (FR-δ);-   [100] a method of preparing the composition of any of [55]-[74],    comprising the steps of: forming a mixture comprising: liposomal    components and alpha polyglutamated Antifolate in a solution;    processing the mixture to form liposomes entrapping and/or    encapsulating alpha polyglutamated Antifolate; and providing a    targeting moiety on a surface of the liposomes, the targeting moiety    having specific affinity for at least one of folate receptor alpha    (FR-α), folate receptor beta (FR-β) and folate receptor delta    (FR-δ);-   [101] the method of [100], wherein the processing step comprises    homogenizing the mixture to form liposomes in the solution;-   [102] the method according to any of [99] to [101], wherein the    processing step includes one or more steps of: thin film hydration,    extrusion, in-line mixing, ethanol injection technique,    freezing-and-thawing technique, reverse-phase evaporation, dynamic    high pressure microfluidization, microfluidic mixing, double    emulsion, freeze-dried double emulsion, 3D printing, membrane    contactor method, and stirring;-   [103] the method according to any of [99] to [102], wherein said    processing step includes one or more steps of modifying the size of    the liposomes by one or more of steps of extrusion, high-pressure    microfluidization, and/or sonication; and/or-   [104] the method of any of [96] to [103], wherein at least 1% of the    starting material of alpha polyglutamated Antifolate is encapsulated    or entrapped in the Lp-αPANTIFOL.

In some embodiments, the disclosure provides an alpha polyglutamatedAntifolate (αPANTIFOL) composition wherein at least one of the glutamylresidues of the alpha polyglutamated Antifolate is linked by its alphacarboxyl group. In some embodiments, the αPANTIFOL contains 2-20, 2-15,2-10, 2-5, or more than 5, glutamyl groups (including the glutamyl groupof the Antifolate). In some embodiments, the alpha polyglutamatedAntifolate is selected from: (a) AG2034, piritrexim, pralatrexate,GW1843, Antifolate, and LY309887; or (b) PMX, MTX, RTX, and LMX, or astereoisomer thereof. In some embodiments, the alpha polyglutamatedAntifolate is selected from: LV (etoposide), L-leucovorin(L-5-formyltetrahydrofolate); 5-CH3-THF, 5-methyltetrahydrofolate; FA,folic acid; PteGlu, pteroyl glutamate (FA); MTX, methotrexate; 2-dMTX,2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin;2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-Ndelta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, Nalpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, Nalpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dPteOrn, Nalpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoicacid; 5-dH4PteOro, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,N10-propargyl-5,8-dideazafolic acid; ICI-198,583,2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583; 7-CH3-ICI-198,583,7-methyl-ICI-198,583; ZD1694,N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamicacid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,(S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl]-glutaricacid; LY231514,N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamicacid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaaisofolicacid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid;N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337,3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline; andAG377, 2,4-diamino-6[N-(4-(phenylsulfonyl) benzyl)ethyl)amino]quinazoline; or a stereoisomer thereof. In some embodiments,the alpha polyglutamated Antifolate is selected from: methotrexate,raltitrexed, plevitrexed, pemetrexed, lometrexol (LMX;5,10-dideazatetrahydrofolic acid), a cyclopenta[g]quinazoline with adipeptide ligand, CB3717, CB300945, or a stereoisomer thereof, such as6-R,S-BGC 945 (ONX-0801), CB300638, and BW1843U89. In some embodiments,the αPANTIFOL comprises two or more glutamyl groups in the L-form. Inother embodiments, the αPANTIFOL comprises a glutamyl group in theD-form. In further embodiments, the αPANTIFOL comprises a glutamyl groupin the D-form and two or more glutamyl groups in the L-form. Inadditional embodiments, the αPANTIFOL comprises two or more glutamylgroups that have a gamma linkage.

In one embodiment, the αPANTIFOL composition contains a chain of 3glutamyl groups attached to the glutamyl group in the Antifolate (i.e.,a tetraglutamated Antifolate). In some embodiments, the αPANTIFOL is apolyglutamate of an Antifolate listed in [2] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [3] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[4] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [5] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the tetraglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In other embodiments, the tetraglutamated Antifolatecomprises a glutamyl group in the D-form. In some embodiments, thetetraglutamated Antifolate comprises two or more glutamyl groups in theD-form. In further embodiments, the tetraglutamated Antifolate comprisesa glutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the tetraglutamated Antifolate comprisesone, two, or three, glutamyl groups in the D-form and three, two, orone, glutamyl groups in the L-form, respectively. In additionalembodiments, the alpha tetraglutamated Antifolate comprises two or moreglutamyl groups that have a gamma linkage.

In one embodiment, the αPANTIFOL composition contains a chain of 4glutamyl groups attached to the glutamyl group in the Antifolate (i.e.,a pentaglutamated Antifolate). In some embodiments, the αPANTIFOL is apolyglutamate of an Antifolate listed in [2] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [3] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[4] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [5] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the pentaglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In other embodiments, the pentaglutamated Antifolatecomprises a glutamyl group in the D-form. In some embodiments, thepentaglutamated Antifolate comprises two or more glutamyl groups in theD-form. In further embodiments, the pentaglutamated Antifolate comprisesa glutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the pentaglutamated Antifolate comprisesone, two, three, or four, glutamyl groups in the D-form and four, three,two, or one, glutamyl groups in the L-form, respectively. In additionalembodiments, the alpha pentaglutamated Antifolate comprises two or moreglutamyl groups that have a gamma linkage.

In one embodiment, the αPANTIFOL composition contains a chain of 5glutamyl groups attached to the glutamyl group in the Antifolate (i.e.,a hexaglutamated Antifolate). In some embodiments, the αPANTIFOL is apolyglutamate of an Antifolate listed in [2] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [3] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[4] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [5] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the hexaglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In other embodiments, the hexaglutamated Antifolatecomprises a glutamyl group in the D-form. In some embodiments, thehexaglutamated Antifolate comprises two or more glutamyl groups in theD-form. In further embodiments, the hexaglutamated Antifolate comprisesa glutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the pentaglutamated Antifolate comprisesone, two, three, four, or five glutamyl groups in the D-form and five,four, three, two, or one, glutamyl groups in the L-form, respectively.In additional embodiments, the alpha hexaglutamated Antifolate comprisestwo or more glutamyl groups that have a gamma linkage.

In additional embodiments, the disclosure provides compositionscontaining delivery vehicles such as liposomes filled with (i.e.,encapsulating) and/or otherwise associated with alpha polyglutamatedAntifolate, and methods of making and using the αPANTIFOLfilled/associated delivery vehicle compositions (DV-αPANTIFOL) todeliver alpha polyglutamated Antifolate to diseased (e.g., cancerous)and/or targeted cells. These compositions have uses that include but arenot limited to treating diseases that include for example,hyperproliferative diseases such as cancer, disorders of the immunesystem such as inflammation and rheumatoid arthritis, and infectiousdiseases such as HIV, malaria, and schistomiasis. The αPANTIFOLfilled/associated delivery vehicle compositions provide improvements tothe efficacy and safety of delivering Antifolate to cancer cells byproviding the preferential delivery of a more cytotoxic payload (e.g.,polyglutamated Antifolate) compared to the cytotoxicity the Antifolateadministered in its monoglutamate state (ANTIFOL). In some embodiments,alpha polyglutamated Antifolate in the DV-αPANTIFOL contains 2-20, 2-15,2-10, 2-5, more than 5, or more than 20, glutamyl groups (including theglutamyl group of the Antifolate). In some embodiments, the deliveryvehicle contains a polyglutamated Antifolate according to any of[1]-[12] of the Brief Summary Section. In some embodiments, the deliveryvehicle contains a polyglutamated Antifolate described in the BriefSummary Section.

In additional embodiments, the disclosure provides a compositioncomprising a liposome encapsulating (filled with) alpha polyglutamatedAntifolate (Lp-αPANTIFOL). In some embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL contains 2-20, 2-15, 2-10, 2-5, or morethan 20, glutamyl groups (including the glutamyl group in theAntifolate). In some embodiments, the alpha polyglutamated Antifolateencapsulated by the liposome is selected from: (a) AG2034, piritrexim,pralatrexate, GW1843, Antifolate, and LY309887; or (b) PMX, MTX, RTX,and LMX, or a stereoisomer thereof. In some embodiments, the alphapolyglutamated Antifolate encapsulated by the liposome is selected from:LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolate); 5-CH3-THF,5-methyltetrahydrofolate; FA, folic acid; PteGlu, pteroyl glutamate(FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX,2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT;2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-Ndelta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, Nalpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, Nalpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dPteOrn, Nalpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoicacid; 5-dH4PteOro, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,N10-propargyl-5,8-dideazafolic acid; ICI-198,583,2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;4-H-TCI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583; 7-CH3-ICI-198,583,7-methyl-ICI-198,583; ZD1694,N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamicacid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,(S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaricacid; LY231514,N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamicacid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaaisofolicacid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid;N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337,3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline; andAG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or a stereoisomer thereof. In someembodiments, the alpha polyglutamated Antifolate encapsulated by theliposome is selected from: methotrexate, raltitrexed, plevitrexed,pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), acyclopenta[g]quinazoline with a dipeptide ligand, CB3717, CB300945, or astereoisomer thereof, such as 6-R,S-BGC 945 (ONX-0801), CB300638, andBW1843U89. In some embodiments, the alpha polyglutamated Antifolate inthe Lp-αPANTIFOL comprises two or more glutamyl groups in the L-form. Inother embodiments, the alpha polyglutamated Antifolate in theLp-αPANTIFOL comprises a glutamyl group in the D-form. In furtherembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the alpha polyglutamated Antifolatein the Lp-αPANTIFOL comprises two or more glutamyl groups that have agamma linkage. In additional embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL comprises one or more glutamyl groupsthat have both an alpha linkage and a gamma linkage. In someembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises 2-10 glutamyl groups that have both an alpha linkage and agamma linkage, or any range therein between. In some embodiments, thepolyglutamate chain of the alpha polyglutamated Antifolate is linear. Insome embodiments, the polyglutamate chain of the alpha polyglutamatedAntifolate is branched.

In one embodiment, the Lp-αPANTIFOL composition comprises an alphapolyglutamated Antifolate that contains a chain of 3 glutamyl groupsattached to the glutamyl group in the Antifolate (i.e., tetraglutamatedAntifolate). In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [2] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[3] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [4] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [5] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate described inthe Brief Summary Section. In some embodiments, the tetraglutamatedAntifolate comprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated Antifolate comprises a glutamyl groupin the D-form. In further embodiments, the tetraglutamated Antifolatecomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In additional embodiments, the tetraglutamated Antifolatecomprises two or more glutamyl groups that have a gamma linkage. In someembodiments, the polyglutamate chain of the alpha polyglutamatedAntifolate is linear. In some embodiments, the polyglutamate chain ofthe alpha polyglutamated Antifolate is branched.

In one embodiment, the Lp-αPANTIFOL composition comprises an alphapolyglutamated Antifolate that contains a chain of 4 glutamyl groupsattached to the glutamyl group in the Antifolate (i.e., pentaglutamatedAntifolate). In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [2] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[3] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [4] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [5] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate described inthe Brief Summary Section. In some embodiments, the pentaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. In otherembodiments, the pentaglutamated Antifolate comprises a glutamyl groupin the D-form. In further embodiments, the pentaglutamated Antifolatecomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In additional embodiments, the pentaglutamated Antifolatecomprises two or more glutamyl groups that have a gamma linkage. In someembodiments, the polyglutamate chain of the alpha polyglutamatedAntifolate is linear. In some embodiments, the polyglutamate chain ofthe alpha polyglutamated Antifolate is branched.

In one embodiment, the Lp-αPANTIFOL composition comprises an alphapolyglutamated Antifolate that contains a chain of 5 glutamyl groupsattached to the glutamyl group in the Antifolate (i.e., hexaglutamatedAntifolate). In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [2] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[3] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [4] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [5] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate described inthe Brief Summary Section. In some embodiments, the hexaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. In otherembodiments, the hexaglutamated Antifolate comprises a glutamyl group inthe D-form. In some embodiments, the hexaglutamated Antifolate comprisestwo or more glutamyl groups in the D-form. In further embodiments, thehexaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepentaglutamated Antifolate comprises one, two, three, four, or fiveglutamyl groups in the D-form and five, four, three, two, or one,glutamyl groups in the L-form, respectively. In additional embodiments,the hexaglutamated Antifolate comprises two or more glutamyl groups thathave a gamma linkage. In some embodiments, the polyglutamate chain ofthe alpha polyglutamated Antifolate is linear. In some embodiments, thepolyglutamate chain of the alpha polyglutamated Antifolate is branched.

In some embodiments, the Lp-αPANTIFOL composition is cationic. In someembodiments, the Lp-αPANTIFOL liposome is cationic and has a diameter inthe range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nmto 150 nm, or any range therein between. In further embodiments, theLp-αPANTIFOL liposome is cationic and the composition has a diameter inthe range of 80 nm to 120 nm, or any range therein between. In someembodiments, the cationic Lp-αPANTIFOL composition comprises at least1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, w/w of the alpha polyglutamated Antifolate. Insome embodiments, during the process of preparing the Lp-αPANTIFOL, atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, of the starting material of alphapolyglutamated Antifolate is encapsulated (entrapped) in the cationicLp-αPANTIFOL. In additional embodiments, the alpha polyglutamatedAntifolate encapsulated by the liposome is in a HEPES buffered solutionwithin the liposome.

In other embodiments, Lp-αPANTIFOL composition is anionic or neutral. Insome embodiments, the Lp-αPANTIFOL composition is cationic. In someembodiments, the Lp-αPANTIFOL liposome is anionic or neutral and has adiameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175nm, or 50 nm to 150 nm, or any range therein between. In furtherembodiments, the Lp-αPANTIFOL liposome is anionic or neutral and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-αPANTIFOL liposome isanionic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between.In further embodiments, the Lp-αPANTIFOL liposome is anionic and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-αPANTIFOL liposome isneutral and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between.In further embodiments, the Lp-αPANTIFOL liposome is neutral and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the anionic or neutralLp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/wof the alpha polyglutamated Antifolate. In some embodiments, during theprocess of preparing the Lp-αPANTIFOL, at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%,of the starting material of alpha polyglutamated Antifolate isencapsulated (entrapped) in the anionic or neutral Lp-αPANTIFOL. In someembodiments, the anionic or neutral Lp-αPANTIFOL composition comprisesat least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, w/w of the alpha tetraglutamatedAntifolate. In some embodiments, the anionic or neutral Lp-αPANTIFOLcomposition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphapentaglutamated Antifolate. In some embodiments, the anionic or neutralLp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/wof the alpha hexaglutamated Antifolate. In additional embodiments, thealpha polyglutamated Antifolate encapsulated by the liposome is in aHEPES buffered solution within the liposome.

In additional embodiments, the liposomal alpha polyglutamated Antifolatecomposition is pegylated (PLp-αPANTIFOL).

In some embodiments, the liposomal alpha polyglutamated Antifolatecomposition is non-targeted (NTLp-αPANTIFOL). That is, theNTLp-αPANTIFOL composition does not have specific affinity towards anepitope (e.g., an epitope on a surface antigen) expressed on the surfaceof a target cell of interest. In some embodiments, the NTLp-αPANTIFOLcomposition does not comprise a targeting moiety. In furtherembodiments, the non-targeted liposomal alpha polyglutamated Antifolatecomposition is pegylated (NTPLp-αPANTIFOL).

In other embodiments, the liposomal alpha polyglutamated Antifolatecomposition is targeted (TLp-αPANTIFOL). That is, the TLp-αPANTIFOLcomposition contains a targeting moiety that has specific affinity foran epitope (surface antigen) on a target cell of interest. In someembodiments, the targeting moiety of the TLp-αPANTIFOL or TPLp-αPANTIFOLis not attached to the liposome through a covalent bond. In otherembodiments, the targeting moiety of the TLp-αPANTIFOL or TPLp-αPANTIFOLis attached to one or both of a PEG and the exterior of the liposome. Insome embodiments, the targeting moiety of the TLp-αPANTIFOL orTPLp-αPANTIFOL is attached to the liposome through a covalent bond.Functions of the targeting moiety of the TLp-αPANTIFOL and/orTPLp-αPANTIFOL compositions include but are not limited to, targetingthe liposome to the target cell of interest in vivo or in vitro;interacting with the surface antigen for which the targeting moiety hasspecific affinity, and delivering the liposome payload (αPANTIFOL) intothe cell. Suitable targeting moieties are known in the art and include,but are not limited to, antibodies, antigen-binding antibody fragments,scaffold proteins, polypeptides, and peptides. In some embodiments, thetargeting moiety is a polypeptide. In further embodiments, the targetingmoiety is a polypeptide that comprises at least 3, 5, 10, 15, 20, 30,40, 50, or 100, amino acid residues.

Targeted liposomal alpha polyglutamated Antifolate compositions(TLp-αPANTIFOL and TPLp-αPANTIFOL) provide further improvements over theefficacy and safety profile of the Antifolate, by specificallydelivering alpha polyglutamated (e.g., tetraglutamated, pentaglutamatedand hexaglutamated) Antifolate to target cells such as cancer cells. Infurther embodiments, the targeted liposomal alpha polyglutamatedAntifolate composition is pegylated (TPLp-αPANTIFOL). Function of thetargeting moiety of the TLp-αPANTIFOL and/or TPLp-αPANTIFOL compositionsinclude but are not limited to, targeting the liposome to the targetcell of interest in vivo or in vitro; interacting with the surfaceantigen for which the targeting moiety has specific affinity, anddelivering the liposome payload (αPANTIFOL) into the cell.

Suitable targeting moieties are known in the art and include, but arenot limited to, antibodies, antigen-binding antibody fragments, scaffoldproteins, polypeptides, and peptides. In some embodiments, the targetingmoiety is a polypeptide. In further embodiments, the targeting moiety isa polypeptide that comprises at least 3, 5, 10, 15, 20, 30, 40, 50, or100, amino acid residues. In some embodiments, the targeting moiety isan antibody or an antigen-binding antibody fragment. In furtherembodiments, the targeting moiety comprises one or more of an antibody,a humanized antibody, an antigen binding fragment of an antibody, asingle chain antibody, a single-domain antibody, a bi-specific antibody,a synthetic antibody, a pegylated antibody, and a multimeric antibody.In some embodiments, the targeting moiety of the TLp-αPANTIFOL orTPLp-αPANTIFOL has specific affinity for an epitope that ispreferentially expressed on a target cell such as a tumor cell, comparedto normal or non-tumor cells. In some embodiments, the targeting moietyhas specific affinity for an epitope on a tumor cell surface antigenthat is present on a tumor cell but absent or inaccessible on anon-tumor cell. In some embodiments, the targeting moiety binds anepitope of interest with an equilibrium dissociation constant (Kd) in arange of 0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis.

In particular embodiments, the TLp-αPANTIFOL or TPLp-αPANTIFOL targetingmoiety comprises a polypeptide that specifically binds a folatereceptor. In some embodiments, the targeting moiety is an antibody or anantigen-binding antibody fragment. In some embodiments, the folatereceptor bound by the targeting moiety is one or more folate receptorsselected from: folate receptor alpha (FR-α, FOLR1), folate receptor beta(FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In someembodiments, the folate receptor bound by the targeting moiety is folatereceptor alpha (FR-α). In some embodiments, the folate receptor bound bythe targeting moiety is folate receptor beta (FR-β). In someembodiments, the targeting moiety specifically binds FR-α and FR-β.

In additional embodiments, the liposome αPANTIFOL composition comprisesone or more of an immunostimulatory agent, a detectable marker, and amaleimide, disposed on at least one of the PEG and the exterior of theliposome. In some embodiments, the liposome αPANTIFOL composition (e.g.,Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL) is cationic. In other embodiments, theliposome αPANTIFOL composition (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) isanionic or neutral. In additional embodiments, the liposome of theliposome αPANTIFOL composition (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) has adiameter in the range of 20 nm to 200 nm, or any range therein between.In further embodiments, the liposome of the liposome αPANTIFOLcomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the liposome αPANTIFOL compositionis pegylated (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL).In some embodiments, the liposome αPANTIFOL composition comprises atargeting moiety (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In furtherembodiments, the liposome αPANTIFOL composition is pegylated andtargeted (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomeαPANTIFOL composition comprises an alpha polyglutamated Antifolate thatcontains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome αPANTIFOL composition comprises an alphatetraglutamated Antifolate. In some embodiments, the liposome αPANTIFOLcomposition comprises an alpha pentaglutamated Antifolate. In otherembodiments, the liposome αPANTIFOL composition comprises an alphahexaglutamated Antifolate. In some embodiments, the αPANTIFOL is apolyglutamate of an Antifolate listed in [2] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamate of anAntifolate listed in [3] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamate of an Antifolate listed in[4] of the Brief Summary Section. In some embodiments, the αPANTIFOL isa polyglutamate of an Antifolate listed in [5] of the Brief SummarySection. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section.

In some embodiments, the liposome compositions comprises an alphapolyglutamated Antifolate that contains 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thealpha polyglutamated Antifolate. In some embodiments, the Lp-αPANTIFOLcomposition comprises an alpha polyglutamated Antifolate that contains4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups and 1%-98.5% w/w ofthe alpha polyglutamated Antifolate. In some embodiments, the liposomescomprise alpha polyglutamated Antifolate that contains 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups and wherein during the process ofpreparing the Lp-αPANTIFOL, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of thestarting material of alpha polyglutamated Antifolate is encapsulated(entrapped) in the Lp-αPANTIFOL. In some embodiments, the liposomecomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, theliposome composition comprises a liposome according to any of [13]-[72]of the Brief Summary Section. In some embodiments, the compositioncomprises an alpha polyglutamated Antifolate described in the BriefSummary Section.

In additional embodiments, the liposome αPANTIFOL composition (i.e.,Lp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL or TPLp-αPANTIFOL) comprises at least 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, liposomeentrapped alpha polyglutamated Antifolate. In some embodiments, theliposome αPANTIFOL composition comprises 1%-98.5% liposome entrappedalpha polyglutamated Antifolate. In additional embodiments, the liposomeαPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, liposome entrappedalpha polyglutamated Antifolate that contains 4, 5, 6, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the liposomeαPANTIFOL composition comprises 1%-98.5% liposome entrapped alphapolyglutamated Antifolate that contains 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups. In some embodiments, the liposome αPANTIFOLcomposition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, liposome entrapped alphatetraglutamated Antifolate. In some embodiments, the liposomecomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, theliposome composition comprises a liposome according to any of [13]-[72]of the Brief Summary Section. In some embodiments, the liposomecomposition comprises an alpha polyglutamated Antifolate described inthe Brief Summary Section.

In some embodiments, the liposome compositions comprise of alphatetraglutamated Antifolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w ofthe alpha tetraglutamated Antifolate. In some embodiments, theLp-αPANTIFOL composition comprises an alpha tetraglutamated Antifolateand 1%-98.5% w/w of the alpha tetraglutamated Antifolate. In someembodiments, the liposomes comprise alpha tetraglutamated Antifolate andwherein during the process of preparing the Lp-αPANTIFOL, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75% of the starting material of alpha tetraglutamatedAntifolate is encapsulated (entrapped) in the Lp-αPANTIFOL. In someembodiments, the liposome composition comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Brief Summary Section. Insome embodiments, the liposome composition comprises a liposomeaccording to any of [13]-[72] of the Brief Summary Section. In someembodiments, the liposome composition comprises an alpha polyglutamatedAntifolate described in the Brief Summary Section.

In some embodiments, the liposome compositions comprise of alphapentaglutamated Antifolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w ofthe alpha pentaglutamated Antifolate. In some embodiments, theLp-αPANTIFOL composition comprises an alpha pentaglutamated Antifolateand 1%-98.5% w/w of the alpha pentaglutamated Antifolate. In someembodiments, the liposomes comprise alpha pentaglutamated Antifolate andwherein during the process of preparing the Lp-αPANTIFOL, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75% of the starting material of alpha pentaglutamatedAntifolate is encapsulated (entrapped) in the Lp-αPANTIFOL. In someembodiments, the liposome compositions comprise of alpha hexaglutamatedAntifolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphahexaglutamated Antifolate. In some embodiments, the Lp-αPANTIFOLcomposition comprises an alpha hexaglutamated Antifolate and 1%-98.5%w/w of the alpha hexaglutamated Antifolate. In some embodiments, theliposomes comprise alpha hexaglutamated Antifolate and wherein duringthe process of preparing the Lp-αPANTIFOL, at least 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than75% of the starting material of alpha pentaglutamated Antifolate isencapsulated (entrapped) in the Lp-αPANTIFOL. In some embodiments, theliposome αPANTIFOL composition comprises 1%-98.5% liposome entrappedalpha pentaglutamated Antifolate. In some embodiments, the liposomecomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, theliposome composition comprises a liposome according to any of [13]-[72]of the Brief Summary Section. In some embodiments, the liposomecomposition comprises an alpha polyglutamated Antifolate described inthe Brief Summary Section.

In some embodiments, the liposome αPANTIFOL composition comprises1%-98.5% liposome entrapped alpha tetraglutamated Antifolate In someembodiments, the liposome αPANTIFOL composition comprises at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or75%, liposome entrapped alpha pentaglutamated Antifolate. In someembodiments, the liposome αPANTIFOL composition comprises 1%-98.5%liposome entrapped alpha pentaglutamated Antifolate. In someembodiments, the liposome αPANTIFOL composition comprise at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or75%, liposome entrapped alpha hexaglutamated Antifolate. In someembodiments, the liposome composition comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Brief Summary Section. Insome embodiments, the liposome composition comprises a liposomeaccording to any of [13]-[72] of the Brief Summary Section. In someembodiments, the liposome composition comprises an alpha polyglutamatedAntifolate described in the Brief Summary Section.

Liposomal compositions comprising liposomes encapsulating αPANTIFOL arealso provided. In some embodiments, the liposomal composition comprisesa pegylated αPANTIFOL composition. In some embodiments, the liposomalcomposition comprise a αPANTIFOL composition that is linked to orotherwise associated with a targeting moiety. In further embodiments,the liposomal composition comprises a αPANTIFOL composition that ispegylated and linked to or otherwise associated with a targeting moiety.In some embodiments, the liposomal composition comprises αPANTIFOL thatcontains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposomal composition comprises an alphatetraglutamated Antifolate. In some embodiments, the liposomalcomposition comprises an alpha pentaglutamated Antifolate. In otherembodiments, the liposomal composition comprises an alpha hexaglutamatedAntifolate. In some embodiments, the liposome composition comprises analpha polyglutamated Antifolate according to any of [1]-[12] of theBrief Summary Section. In some embodiments, the liposome compositioncomprises a liposome according to any of [13]-[72] of the Brief SummarySection. In some embodiments, the liposome composition comprises analpha polyglutamated Antifolate described in the Brief Summary Section.

In some embodiments, the liposomal composition comprises a liposomeαPANTIFOL (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL, and TPLp-αPANTIFOL). In someembodiments, the liposome αPANTIFOL is pegylated (e.g., NTPLp-αPANTIFOL,and TPLp-αPANTIFOL). In some embodiments, the pharmaceutical compositioncomprises αPANTIFOL that contains 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the pharmaceutical compositioncomprises alpha tetraglutamated Antifolate. In some embodiments, thepharmaceutical composition comprises alpha pentaglutamated Antifolate.In other embodiments, the pharmaceutical composition comprises alphahexaglutamated Antifolate. In some embodiments, the liposome compositioncomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Brief Summary Section. In some embodiments, the liposomecomposition comprises a liposome according to any of [13]-[72] of theBrief Summary Section. In some embodiments, the liposome compositioncomprises an alpha polyglutamated Antifolate described in the BriefSummary Section. In some embodiments, the liposome αPANTIFOL comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of a target cell of interest such as a cancer cell (e.g.,TLp-αPANTIFOL or TPLp-αPANTIFOL). In further embodiments, the liposomalcomposition comprises a liposome αPANTIFOL that is pegylated and furthercomprises a targeting moiety that has a specific affinity for an epitopeof antigen on the surface of a target cell of interest such as a cancercell (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomalcomposition comprises a liposome αPANTIFOL that is cationic. In otherembodiments, the liposomal composition comprises a liposome αPANTIFOLthat is anionic or neutral. In additional embodiments, the liposomalcomposition comprises a liposome αPANTIFOL that has a diameter in therange of 20 nm to 500 nm, 20 nm to 200 nm, or any range therein between.In further embodiments, the liposome αPANTIFOL has a diameter in therange of 80 nm to 120 nm, or any range therein between.

Pharmaceutical compositions comprising alpha polyglutamated Antifolate(αPANTIFOL) including delivery vehicles such as liposome αPANTIFOL arealso provided. In some embodiments, the pharmaceutical compositioncomprises a pegylated αPANTIFOL composition. In some embodiments, thepharmaceutical composition comprise a αPANTIFOL composition that islinked to or otherwise associated with a targeting moiety. In furtherembodiments, the pharmaceutical composition comprise a αPANTIFOLcomposition that is pegylated and linked to or otherwise associated witha targeting moiety. In some embodiments, the pharmaceutical compositioncomprises αPANTIFOL that contains 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the pharmaceutical compositioncomprises an alpha tetraglutamated Antifolate. In some embodiments, thepharmaceutical composition comprises an alpha pentaglutamatedAntifolate. In other embodiments, the pharmaceutical compositioncomprises an alpha hexaglutamated Antifolate. In other embodiments, thepharmaceutical composition comprises alpha hexaglutamated Antifolate. Insome embodiments, the composition comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Brief Summary Section. Insome embodiments, the pharmaceutical composition comprises a liposomecomposition according to any of [13]-[74] of the Brief Summary Section.In some embodiments, the pharmaceutical composition comprises an alphapolyglutamated Antifolate described in the Brief Summary Section. Insome embodiments, the pharmaceutical composition comprises apolyglutamated Antifolate described in the Brief Summary Section.

In some embodiments, the pharmaceutical compositions comprise a liposomeαPANTIFOL (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL, and TPLp-αPANTIFOL). In someembodiments, the liposome αPANTIFOL composition is pegylated (e.g.,NTPLp-αPANTIFOL, and TPLp-αPANTIFOL). In some embodiments, the liposomeαPANTIFOL comprises a targeting moiety that has a specific affinity foran epitope of antigen on the surface of a target cell of interest suchas a cancer cell (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In furtherembodiments, the pharmaceutical composition comprises a liposomeαPANTIFOL composition that is pegylated and further comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of a target cell of interest such as a cancer cell (e.g.,TPLp-αPANTIFOL). In some embodiments, the pharmaceutical compositioncomprises a liposome αPANTIFOL that is cationic. In other embodiments,the pharmaceutical composition comprises a liposome αPANTIFOL that isanionic or neutral. In additional embodiments, the pharmaceuticalcomposition comprises a liposome αPANTIFOL that has a diameter in therange of 20 nm to 500 nm or 20 nm to 500 nm, or any range thereinbetween. In further embodiments, the liposome αPANTIFOL composition hasa diameter in the range of 80 nm to 120 nm, or any range thereinbetween. In some embodiments, the pharmaceutical composition comprisesαPANTIFOL that contains 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the pharmaceutical composition comprisesalpha tetraglutamated Antifolate. In some embodiments, thepharmaceutical composition comprises alpha pentaglutamated Antifolate.In some embodiments, the pharmaceutical composition comprises an alphapolyglutamated Antifolate according to any of [1]-[12] of the BriefSummary Section. In some embodiments, the pharmaceutical compositioncomprises a liposome composition according to any of [13]-[74] of theBrief Summary Section. In some embodiments, the pharmaceuticalcomposition comprises an alpha polyglutamated Antifolate described inthe Brief Summary Section.

In additional embodiments, the disclosure provides a method ofmodulating the activation, chemokine production, or metabolic activityof a cell that comprises contacting the cell with a compositioncomprising an alpha polyglutamated Antifolate (αPANTIFOL) composition.In some embodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the cell is an immune cell. In some embodiments, the method is performedin vivo. In other embodiments, the method is performed in vitro. In someembodiments, the αPANTIFOL contains 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPANTIFOL compositioncomprises an alpha tetraglutamated Antifolate. In some embodiments, theαPANTIFOL composition comprises an alpha pentaglutamated Antifolate. Inother embodiments, the αPANTIFOL composition comprises an alphahexaglutamated Antifolate. In some embodiments, the αPANTIFOLcomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, thecomposition comprises a liposome composition according to any of[13]-[72] of the Brief Summary Section. In some embodiments, thecomposition comprises an alpha polyglutamated Antifolate described inthe Brief Summary Section.

In additional embodiments, the disclosure provides a method ofmodulating the activation, chemokine production, or metabolic activityof a cell that comprises contacting the cell with a liposome comprisingan alpha polyglutamated Antifolate (αPANTIFOL) composition. In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the cell is an immune cell. In some embodiments, the method is performedin vivo. In other embodiments, the method is performed in vitro. In someembodiments, the αPANTIFOL contains 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPANTIFOL compositioncomprises an alpha tetraglutamated Antifolate. In some embodiments, theαPANTIFOL composition comprises an alpha pentaglutamated Antifolate. Inother embodiments, the αPANTIFOL composition comprises an alphahexaglutamated Antifolate. In some embodiments, the liposome comprisesan alpha polyglutamated Antifolate according to any of [1]-[12] of theBrief Summary Section. In some embodiments, the liposome is a liposomeaccording to any of [13]-[72] of the Brief Summary Section. In someembodiments, the liposome comprises an alpha polyglutamated Antifolatedescribed in the Brief Summary Section.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a composition comprising analpha polyglutamated Antifolate (αPANTIFOL) composition. In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the hyperproliferative cell is a cancer cell. In further embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from: a non-hematologicmalignancy including such as for example, lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In some embodiments, the cancer cell is aprimary cell or a cell from a cell line obtained/derived from a cancerselected from: breast cancer, advanced head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladdercancer, and central nervous system (CNS) lymphoma. In some embodiments,the method is performed in vivo. In other embodiments, the method isperformed in vitro. In some embodiments, the αPANTIFOL contains 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theαPANTIFOL composition comprises an alpha tetraglutamated Antifolate. Insome embodiments, the αPANTIFOL composition comprises an alphapentaglutamated Antifolate. In other embodiments, the αPANTIFOLcomposition comprises an alpha hexaglutamated Antifolate. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, theαPANTIFOL is a polyglutamated Antifolate described in the Brief SummarySection. In some embodiments, the αPANTIFOL composition comprises aliposome according to any of [13]-[72] of the Brief Summary Section.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containing alphapolyglutamated Antifolate (e.g. an Lp-αPANTIFOL such as, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL). Insome embodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the contacted hyperproliferative cell is a cancer cell. In furtherembodiments, the cancer cell is a primary cell or a cell from a cellline obtained/derived from a cancer selected from: a non-hematologicmalignancy including such as for example, lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In some embodiments, the cell is a primarycell or a cell from a cell line obtained/derived from a cancer selectedfrom: breast cancer, advanced head and neck cancer, lung cancer, stomachcancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma)choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer, softtissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer,and central nervous system (CNS) lymphoma. In some embodiments, themethod is performed in vivo. In other embodiments, the method isperformed in vitro. In some embodiments, the liposome contains aαPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the liposome contains alpha tetraglutamatedAntifolate. In some embodiments, the liposome contains alphapentaglutamated Antifolate. In other embodiments, the liposome containsalpha hexaglutamated Antifolate. In some embodiments, the liposomecomprises a polyglutamated Antifolate according to any of [1]-[12] ofthe Brief Summary Section. In some embodiments, the liposome comprises apolyglutamated Antifolate described in the Brief Summary Section. Insome embodiments, the liposomal composition comprises a liposomeaccording to any of [13]-[72] of the Brief Summary Section.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., an antibody immunoconjugate or liposome) comprising alphapolyglutamated Antifolate to a subject having or at risk of havingcancer. In some embodiments, the delivery vehicle is anantibody-containing immunoconjugate (comprising e.g., a full-length IgGantibody, a bispecific antibody, or a scFv). In some embodiments, thedelivery vehicle is a liposome (e.g., an Lp-αPANTIFOL such as,PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, orTPLp-αPANTIFOL). In some embodiments, the administered delivery vehicleis pegylated. In some embodiments, the administered delivery vehicle isnot pegylated. In additional embodiments, the administered deliveryvehicle comprises a targeting moiety that has a specific affinity for anepitope of antigen on the surface of a cancer cell. In additionalembodiments, the delivery vehicle comprises a targeting moiety thatspecifically binds a cell surface antigen selected from: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P-Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, EGFR, IGFR-1, EGFRvIII, CD2, CD3, CD4,CD5, CD6, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30,CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b,CD105, CD133, CD138, cripto, CD38, an EphA receptor, an EphB receptor,EphA2, an integrin (e.g., integrin α_(v)β₃, α_(v)β₅, or α_(v)β₆), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56,CanAg, and CALLA. In some embodiments, the delivery vehicle comprises atargeting moiety that specifically binds a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the targetingmoiety specifically binds a cell surface antigen(s) derived from ordetermined to be expressed on a specific subject's tumor such as aneoantigen. In some embodiments, the targeting moiety is an antibody oran antigen binding antibody fragment. In some embodiments, theadministered delivery vehicle comprises αPANTIFOL containing 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises an alpha tetraglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises an alpha pentaglutamated Antifolate. In other embodiments, theadministered delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises L alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises 2, 3, 4, 5, or more than 5,L-alpha glutamyl groups. In some embodiments, the administered deliveryvehicle comprises D alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises L and D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups and 2, 3,4, 5, or more than 5, D-alpha glutamyl groups. In some embodiments, thecancer is selected from: a non-hematologic malignancy including such asfor example, lung cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dysplasias or dyscrasias. Insome embodiments, the cancer cell is a primary cell or a cell from acell line obtained/derived from a cancer selected from: breast cancer,advanced head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralnervous system (CNS) cancer.

In some embodiments, the contacted cancer cell is a primary cell or acell from a cell line obtained/derived from lung cancer (e.g., NSCLC ormesothelioma). In some embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from breastcancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from colorectal cancer. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from ovarian cancer. In some embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from endometrial cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from pancreatic cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from liver cancer. In some embodiments, the contactedcancer cell is a primary cell or a cell from a cell lineobtained/derived from head and neck cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from osteosarcoma. In some embodiments, theadministered delivery vehicle comprises a polyglutamated Antifolateaccording to any of [1]-[12] of the Brief Summary Section. In someembodiments, the delivery vehicle comprises a polyglutamated Antifolatedescribed in the Brief Summary Section. In some embodiments, theliposomal composition comprises a liposome according to any of [13]-[72]of the Brief Summary Section.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising alpha polyglutamated Antifolate (e.g., an Lp-αPANTIFOL suchas, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, orTPLp-αPANTIFOL) to a subject having or at risk of having cancer. In someembodiments, the liposome is pegylated. In some embodiments, theliposome is not pegylated. In additional embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeof antigen on the surface of a cancer cell. In additional embodiments,the liposome comprises a targeting moiety that specifically binds a cellsurface antigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P-Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, EGFR, IGFR-1, EGFRvIII, CD2, CD3, CD4, CD5, CD6, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30, CD33, CD34,CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD105,CD133, CD138, cripto, CD38, an EphA receptor, an EphB receptor, EphA2,an integrin (e.g., integrin α_(v)β₃, α_(v)β₅, or α_(v)β₆), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56,CanAg, and CALLA. This also includes the use of cancer stem celltargeting moieties such as those targeting CD34, CD133 and CD44, CD138,and CD15. In some embodiments, the liposome comprises a targeting moietythat specifically binds a cell surface antigen(s) derived from ordetermined to be expressed on a specific subject's tumor such as aneoantigen. In some embodiments, the targeting moiety is an antibody oran antigen binding antibody fragment. In some embodiments, the liposomecomprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the liposome comprises an alphatetraglutamated Antifolate. In some embodiments, the liposome comprisesan alpha pentaglutamated Antifolate. In other embodiments, the liposomecomprises an alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises a polyglutamated Antifolate according to any of[1]-[12] of the Brief Summary Section. In some embodiments, theαPANTIFOL is a polyglutamated Antifolate described in the Brief SummarySection. In some embodiments, the liposome is a liposome according toany of [13]-[72] of the Brief Summary Section. In some embodiments, theliposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups.In some embodiments, the liposome comprises D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, the liposomecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the liposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the cancer is selected from: lung (e.g., non-small lungcancer), pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, melanoma, and a hematologic malignancy (e.g., a leukemia orlymphoma). In some embodiments, the cancer is selected from: breastcancer, advanced head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis), bladder cancer, and centralnervous system (CNS) cancer. In some embodiments, the cancer is lungcancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer isbreast cancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the cancer is colorectal cancer. In some embodiments, thecancer is ovarian cancer. In some embodiments, the cancer is endometrialcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition comprisinga liposome that comprises an alpha polyglutamated Antifolate and atargeting moiety that has a specific affinity for an epitope of antigenon the surface of the cancer. In some embodiments, the liposomecomprises a targeting moiety that specifically binds a cell surfaceantigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P-Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, EGFR, IGFR-1, EGFRvII, CD2, CD3, CD4, CD5, CD6, CD1, CD11a,CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD105, CD133, CD138,cripto, CD38, an EphA receptor, an EphB receptor, EphA2, an integrin(e.g., integrin α_(v)β₃, α_(v)β₅, or α_(v)β₆), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56, CanAg, and CALLA.In some embodiments, the administered liposome comprises a targetingmoiety that specifically binds a cell surface antigen(s) derived from,or determined to be expressed on, a specific subject's tumor such as aneoantigen. In some embodiments, the administered liposomal compositioncomprises pegylated liposomes (e.g., TPLp-αPANTIFOL). In someembodiments, the administered liposomal composition comprises liposomesthat are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprise a αPANTIFOL containing 4, 5,6, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,liposomes of the administered liposomal composition comprise alphatetraglutamated Antifolate. In some embodiments, liposomes of theadministered liposomal composition comprise alpha pentaglutamatedAntifolate. In other embodiments, liposomes of the administeredliposomal composition comprise alpha hexaglutamated Antifolate. In someembodiments, the liposome comprises a polyglutamated Antifolateaccording to any of [1]-[12] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate described inthe Brief Summary Section. In some embodiments, the liposome compositioncomprises a liposome according to any of [13]-[72] of the Brief SummarySection. In some embodiments, the liposomal composition is administeredto treat a cancer selected from: lung cancer (e.g., non-small cell),pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma and other plasma cell dysplasias or dyscrasias, and leukemia anda lymphoma and other B cell malignancies. In some embodiments, theliposomal composition is administered to treat a cancer selected from:breast cancer, advanced head and neck cancer, lung cancer, stomachcancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma)choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer, softtissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer,and central nervous system (CNS) cancer. In some embodiments, theliposomal composition is administered to treat lung cancer (e.g., NSCLCor mesothelioma). In some embodiments, the liposomal composition isadministered to treat breast cancer (e.g., HER2++ or triple negativebreast cancer). In some embodiments, the liposomal composition isadministered to treat colorectal cancer. In some embodiments, theliposomal composition is administered to treat ovarian cancer. In someembodiments, the liposomal composition is administered to treatendometrial cancer. In some embodiments, the liposomal composition isadministered to treat pancreatic cancer. In some embodiments, theliposomal composition is administered to treat liver cancer. In someembodiments, the liposomal composition is administered to treat head andneck cancer. In some embodiments, the liposomal composition isadministered to treat osteosarcoma.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) alpha polyglutamatedAntifolate (αPANTIFOL) and (b) a targeting moiety that has specificbinding affinity for a folate receptor. In some embodiments, thetargeting moiety has specific binding affinity for folate receptor alpha(FR-α), folate receptor beta (FR-β), and/or folate receptor delta(FR-δ). In some embodiments, the targeting moiety has a specific bindingaffinity for folate receptor alpha (FR-α) and folate receptor beta(FR-β). In some embodiments, the administered liposomal compositioncomprises pegylated liposomes (e.g., TPLp-αPANTIFOL). In someembodiments, the administered liposomal composition comprises liposomesthat are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprises an αPANTIFOL containing 4,5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,liposomes of the administered liposomal composition comprise alphatetraglutamated Antifolate. In some embodiments, liposomes of theadministered liposomal composition comprise alpha pentaglutamatedAntifolate. In other embodiments, liposomes of the administeredliposomal composition comprises an alpha hexaglutamated Antifolate. Insome embodiments, the liposome comprises a polyglutamated Antifolateaccording to any of [1]-[12] of the Brief Summary Section. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate described inthe Brief Summary Section. In some embodiments, the liposome compositioncomprises a liposome according to any of [13]-[72] of the Brief SummarySection. In some embodiments, the liposomal composition is administeredto treat a cancer selected from: a non-hematologic malignancy includingsuch as for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dysplasias or dyscrasias. Insome embodiments, the liposomal composition is administered to treat acancer selected from: breast cancer, advanced head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladdercancer, and central nervous system (CNS) cancer. In some embodiments,the liposomal composition is administered to treat lung cancer (e.g.,NSCLC or mesothelioma). In some embodiments, the liposomal compositionis administered to treat breast cancer (e.g., HER2++ or triple negativebreast cancer). In some embodiments, the liposomal composition isadministered to treat colorectal cancer. In some embodiments, theliposomal composition is administered to treat ovarian cancer. In someembodiments, the liposomal composition is administered to treatendometrial cancer. In some embodiments, the liposomal composition isadministered to treat pancreatic cancer. In some embodiments, theliposomal composition is administered to treat liver cancer. In someembodiments, the liposomal composition is administered to treat head andneck cancer. In some embodiments, the liposomal composition isadministered to treat osteosarcoma.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain alphapolyglutamated Antifolate (Lp-αPANTIFOL) to a subject that is undergoingor has undergone cancer therapy. In some embodiments, the administeredliposomal composition is a PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL. In some embodiments,liposomes of the administered liposomal composition comprises pegylatedliposomes (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, the administered liposomal composition comprisestargeted liposomes (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In someembodiments, the administered liposomal composition comprises liposomesthat are pegylated and comprise a targeting moiety (e.g.,TPLp-αPANTIFOL). In some embodiments, liposomes of the administeredliposomal composition comprises an alpha polyglutamated Antifolate thatcontains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha tetraglutamated Antifolate. In some embodiments,liposomes of the administered liposomal composition comprise alphapentaglutamated Antifolate. In other embodiments, liposomes of theadministered liposomal composition comprise alpha hexaglutamatedAntifolate. In some embodiments, the liposomal composition comprises apolyglutamated Antifolate according to any of [1]-[12] of the BriefSummary Section. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the liposomal composition comprises a liposome according to any of[13]-[72] of the Brief Summary Section.

In additional embodiments, the disclosure provides a method for treatinga disorder of the immune system that comprises administering aneffective amount of a liposomal composition comprising liposomes thatcontain alpha polyglutamated Antifolate (e.g., Lp-αPANTIFOL,PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL orTPLp-αPANTIFOL) to a subject having or at risk of having a disorder ofthe immune system. In some embodiments, the liposomal composition isadministered to treat an autoimmune disease. In a further embodiment,the liposomal composition is administered to treat rheumatoid arthritis.In another embodiment, the liposomal composition is administered totreat inflammation. In some embodiments, the disorder of the immunesystem is selected from: inflammation (e.g., acute and chronic),systemic inflammation, rheumatoid arthritis, inflammatory bowel disease(IBD), Crohn disease, dermatomyositis/polymyositis, systemic lupuserythematosus, Takayasu, and psoriasis. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,the administered liposomal composition comprises targeted liposomes(e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL) that contain a targeting moietyhaving a specific affinity for a surface antigen on a target cell ofinterest (e.g., an immune cell). In further embodiments, theadministered liposomal composition comprises liposomes that arepegylated and comprise a targeting moiety (e.g., TPLp-αPANTIFOL). Insome embodiments, liposomes of the administered liposomal compositioncomprise alpha polyglutamated Antifolate that contains 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, liposomes ofthe administered liposomal composition comprise alpha tetraglutamatedAntifolate. In some embodiments, liposomes of the administered liposomalcomposition comprise alpha pentaglutamated Antifolate. In otherembodiments, liposomes of the administered liposomal compositioncomprise alpha hexaglutamated Antifolate. In some embodiments, theadministered liposomal composition comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Brief Summary Section. Insome embodiments, the αPANTIFOL is a polyglutamated Antifolate describedin the Brief Summary Section. In some embodiments, the liposomalcomposition comprises a liposome according to any of [13]-[72] of theBrief Summary Section.

In additional embodiments, the disclosure provides a method for treatingan autoimmune disease that comprises administering an effective amountof a liposomal composition comprising liposomes that contain alphapolyglutamated Antifolate (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) to asubject having or at risk of having an autoimmune disease. In someembodiments, the autoimmune disease is rheumatoid arthritis. In someembodiments, the autoimmune disease is selected from: inflammatory boweldisease (IBD), Crohn disease, systemic lupus erythematosus, andpsoriasis. In some embodiments, the autoimmune disease is a disease ordisorder selected from: Addison's disease, alopecia areata, ankylosingspondylitis, autoimmune hepatitis, autoimmune parotitis, diabetes (TypeI), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, and ulcerative colitis. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,the administered liposomal composition comprises targeted liposomes(e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL) that contain a targeting moietyhaving a specific affinity for a surface antigen on a target cell ofinterest (e.g., an immune cell). In further embodiments, theadministered liposomal composition comprises liposomes that arepegylated and comprise a targeting moiety (e.g., TPLp-αPANTIFOL). Insome embodiments, liposomes of the administered liposomal compositioncomprise alpha polyglutamated Antifolate that contains 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, liposomes ofthe administered liposomal composition comprise alpha tetraglutamatedAntifolate. In some embodiments, liposomes of the administered liposomalcomposition comprise alpha pentaglutamated Antifolate. In otherembodiments, liposomes of the administered liposomal compositioncomprise alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises a polyglutamated Antifolate according to any of[1]-[12] of the Brief Summary Section. In some embodiments, theαPANTIFOL is a polyglutamated Antifolate described in the Brief SummarySection. In some embodiments, the liposome composition comprises aliposome according to any of [13]-[72] of the Brief Summary Section.

In additional embodiments, the disclosure provides a method for treatingan inflammatory disorder that comprises administering an effectiveamount of a liposomal composition comprising liposomes that containalpha polyglutamated Antifolate (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) to asubject having or at risk of having an inflammatory disorder. In someembodiments, the inflammatory disorder is a disorder selected from:acute inflammation, chronic inflammation, systemic inflammation,rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease,dermatomyositis/polymyositis, and systemic lupus erythematosus. In someembodiments, the inflammatory disorder is a disorder selected from: arheumatoid disease or other arthritic disease (e.g., acute arthritis,acute gouty arthritis, bacterial arthritis, chronic inflammatoryarthritis, degenerative arthritis (osteoarthritis), infectiousarthritis, juvenile arthritis, mycotic arthritis, neuropathic arthritis,polyarthritis, proliferative arthritis, psoriatic arthritis, venerealarthritis, viral arthritis), fibrositis, pelvic inflammatory disease,acne, psoriasis, actinomycosis, dysentery, biliary cirrhosis, Lymedisease, heat rash, Stevens-Johnson syndrome, mumps, pemphigus vulgaris,and blastomycosis. In some embodiments, the inflammatory disorder is aninflammatory bowel disease. Inflammatory bowel diseases are chronicinflammatory diseases of the gastrointestinal tract which include,without limitation, Crohn's disease, ulcerative colitis, andindeterminate colitis. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., PLp-αPANTIFOL,NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, theadministered liposomal composition comprises targeted liposomes (e.g.,TLp-αPANTIFOL or TPLp-αPANTIFOL) that contain a targeting moiety havinga specific affinity for a surface antigen on a target cell of interest(e.g., an immune cell). In further embodiments, the administeredliposomal composition comprises liposomes that are pegylated andcomprise a targeting moiety (e.g., TPLp-αPANTIFOL). In some embodiments,liposomes of the administered liposomal composition comprise alphapentaglutamated Antifolate that contains 4, 5, 6, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, liposomes of theadministered liposomal composition comprise alpha tetraglutamatedAntifolate. In some embodiments, liposomes of the administered liposomalcomposition comprise alpha pentaglutamated Antifolate. In otherembodiments, liposomes of the administered liposomal compositioncomprise alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises a polyglutamated Antifolate according to any of[1]-[12] of the Brief Summary Section. In some embodiments, theαPANTIFOL is a polyglutamated Antifolate described in the Brief SummarySection. In some embodiments, the liposome composition comprises aliposome according to any of [13]-[72] of the Brief Summary Section.

The disclosure also provides a method of delivering alpha polyglutamatedAntifolate to a site of inflammation in a subject that comprises:administering to the subject having the inflammation, a compositioncomprising alpha polyglutamated Antifolate (L-αPANTIFOL) and a targetingmoiety that has a specific binding affinity for an epitope on a surfaceantigen on a cell that is located at, or otherwise influences theinflammation (e.g., via proinflammatory cytokine production). In someembodiments, the administered targeting moiety is associated with adelivery vehicle. In some embodiments, the delivery vehicle is anantibody or an antigen binding fragment of an antibody. In furtherembodiments, the delivery vehicle is a liposome. In further embodiments,the antibody, antigen-binding antibody fragment, or liposome ispegylated liposomes (e.g., TPLp-αPANTIFOL). In some embodiments, theadministered composition comprises an alpha polyglutamated Antifolatethat contains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. Insome embodiments, the administered composition comprises an alphatetraglutamated Antifolate. In some embodiments, the administeredcomposition comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered composition comprises an alphahexaglutamated Antifolate. In some embodiments, the αPANTIFOL is apolyglutamated Antifolate according to any of [1]-[12] of the BriefSummary Section. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the delivery vehicle is a liposome according to any of [13]-[72] of theBrief Summary Section.

The disclosure also provides a method of delivering alpha polyglutamatedAntifolate to a tumor cancer cell that comprises: administering to asubject having the tumor, a composition comprising alpha polyglutamatedAntifolate (L-αPANTIFOL) and a targeting moiety that has a specificbinding affinity for an epitope on a surface antigen on the tumor cellor cancer cell. In some embodiments, the administered targeting moietyis associated with a delivery vehicle. In some embodiments, the deliveryvehicle is an antibody or an antigen binding fragment of an antibody. Infurther embodiments, the delivery vehicle is a liposome. In furtherembodiments, the antibody, antigen-binding antibody fragment, orliposome is pegylated liposomes (e.g., TPLp-αPANTIFOL). In someembodiments, the administered composition comprises an alphapolyglutamated Antifolate that contains 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups. In some embodiments, the administered compositioncomprises an alpha tetraglutamated Antifolate. In some embodiments, theadministered composition comprises an alpha pentaglutamated Antifolate.In other embodiments, the administered composition comprises an alphahexaglutamated Antifolate. In some embodiments, the αPANTIFOL is apolyglutamated Antifolate according to any of [1]-[12] of the BriefSummary Section. In some embodiments, the αPANTIFOL is a polyglutamatedAntifolate described in the Brief Summary Section. In some embodiments,the delivery vehicle is a liposome according to any of [13]-[72] of theBrief Summary Section.

In additional embodiments, the disclosure provides a method of preparinga liposomal composition that comprises a liposomal alpha polyglutamatedAntifolate (αPANTIFOL) composition, the method comprising: forming amixture comprising: liposomal components and α polyglutamated Antifolatein solution; homogenizing the mixture to form liposomes in the solution;and processing the mixture to form liposomes containing polyglutamatedAntifolate. In some embodiments, the alpha polyglutamated Antifolatecontains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the polyglutamated Antifolate composition comprises analpha tetraglutamated Antifolate. In some embodiments, thepolyglutamated Antifolate composition comprises an alpha pentaglutamatedAntifolate. In other embodiments, the polyglutamated Antifolatecomposition comprises an alpha hexaglutamated Antifolate. In someembodiments, the αPANTIFOL is a polyglutamated Antifolate according toany of [1]-[12] of the Brief Summary Section. In some embodiments, theαPANTIFOL is a polyglutamated Antifolate described in the Brief SummarySection. In some embodiments, the liposomal composition comprises aliposome according to any of [13]-[72] of the Brief Summary Section.

In one embodiment, the disclosure provides a kit comprising anAntifolate alpha polyglutamate composition and/or αPANTIFOL deliveryvehicles such as liposomes containing αPANTIFOL and αPANTIFOLimmunoconjugates (e.g., ADCs) described herein.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1R show chemical formulas of the Antifolate pemetrexed (FIG.1A), exemplary alpha pemetrexed polyglutamates, alpha pemetrexeddiglutamate (FIG. 1B), alpha pemetrexed triglutamate (FIGS. 1C and 1D),alpha pemetrexed tetraglutamate (FIGS. 1E and 1F), alpha pemetrexedpentaglutamates (FIGS. 1G and 1H), alpha pemetrexed hexaglutamates(FIGS. 1I and 1J), alpha pemetrexed heptaglutamate (FIGS. 1K and 1L),and alpha pemetrexed octaglutamates (FIGS. 1M and 1N). FIGS. 1O-1Rpresent depictions of exemplary branched alpha pemetrexed polyglutamatestructures, including a branched polyglutamate having a gamma glutamylbackbone and alpha glutamyl branches (FIG. 1P) and a branchedpolyglutamate having an alpha glutamyl backbone and gamma glutamylbranches (FIGS. 1Q and 1R).

FIG. 2 presents the relative potency of liposomal pemetrexed alpha-Lhexaglutamate (liposomal aG6) and its mirror image, liposomal alpha-Dhexaglutamate (liposomal aDG6) relative to pemetrexed following exposureof the cancer cell lines SW620 (CRC), HT-29 (colon cancer), HCC1806(triple negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC,adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype), over48 hours.

FIG. 3 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the NCI H2342 non-small cell lung cancer (NSCLC),adenocarcinoma subtype depicted as the percentage of viable cells after48 hours of treatment. Folate receptor alpha targeted liposomescontaining alpha polyglutamated pemetrexed are expected to also besuccessful in targeting and reducing the viability of NC H2342 non-smallcell lung cancer cells.

FIG. 4 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the HT-29 (colon cancer) at 48 hours. Folate receptoralpha targeted liposomes containing alpha polyglutamated pemetrexed areexpected to also be successful in targeting and reducing the viabilityof HT-29 (colon cancer) cells.

FIG. 5 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of liposomal pemetrexed alpha-Lhexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate(Lps Hexa aDG6), and to pemetrexed over 48 hours.

FIG. 6 presents the treatment effect on OAW28 ovarian cancer cellsfollowing exposure of liposomal pemetrexed alpha-L hexaglutamate (LpsHexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6),and to pemetrexed over 48 hours.

FIG. 7 presents the treatment effect on H292 non-small cell lung cancercells following exposure of liposomal pemetrexed alpha-L hexaglutamate(Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps HexaaDG6), as compared to pemetrexed over 48 hours.

FIG. 8 presents the treatment effect on H292 non-small cell lung cancercells following exposure of various dose levels ranging from 16 to 128nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6),liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), andpemetrexed over 48 hours. At each of the tested dose ranges, theliposomal pemetrexed aG6 formulation is superior to inhibiting H292non-small cell lung cancer cells compared to pemetrexed.

FIG. 9 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of various dose levels ranging from 16to 128 nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6),liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), andpemetrexed over 48 hours. At each of the tested doses, the liposomalpemetrexed aG6 formulation is superior to pemetrexed in inhibitingHCC1806 triple negative breast cancer cells.

FIG. 10 presents the treatment effect on OAW28 ovarian cancer cells ofliposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomalalpha-D hexaglutamate (Liposomal aDG6), and pemetrexed followingexposure over 48 hours following exposure over a range ofconcentrations. At the dose of 128 nM, pemetrexed appears to moreeffective than the Liposomal pemetrexed aG6 liposomal formulation,whereas the liposomal formulation at the dose of 32 nM and 64 nM has abetter treatment effect than pemetrexed; at 16 nM the Liposomalpemetrexed aG6 treatment effect is similar in to pemetrexed.

FIG. 11 shows the toxicity of liposomal pemetrexed alpha-L hexaglutamate(Liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (LiposomalaDG6), and pemetrexed on differentiating human neutrophils at 64 nM, 128nM, and 264 nM. The figure demonstrates that liposomal pemetrexed aG6 issignificantly less toxic to differentiating human neutrophils thanpemetrexed.

FIG. 12 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), andpemetrexed on neutrophils (differentiated from CD34+ cells) followingexposure of various dose levels ranging from 16 to 128 nM of thecorresponding agent over 48 hours.

FIG. 13 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomalaDG6), and pemetrexed on AML12 liver cells following exposure over 48hours at 16 nM, 32 nM, and 64 nM, and 128 nM of the corresponding agent.Strikingly, there does not appear to be any toxicity to the AML12 livercells following treatment with a liposomal pemetrexed aG6 at any of theliposomal agents at the dose levels tested. In contrast, pemetrexedtreatment results in a reduction in the AML12 liver cell counts ofapproximately 40% at all doses studied.

FIG. 14 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomalaDG6), and pemetrexed on CCD841 colon epithelium cells followingexposure over 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM, of thecorresponding agent. At all of the concentrations tested, pemetrexedleads to approximately a ≥50% decrease in the number of CCD841 colonepithelium cells compared to approximately a 20% or less decrease incell number after treatment with each of the liposome compositionstested.

FIG. 15 depicts the structure of polyglutamate antifolate, cisplatin(CDDP) and two potential aG6-Cisplatin complexes. The pH dependentformation of the interstrand and/or instrastrand coordination betweenthe carboxyl groups of the polyglutamated Antifolate and cisplatin islikely to disassemble into individual molecules of aG6 and cisplatinupon encountering acidic pH of lysosomes (pH 4-5) and presence ofchloride ions inside the cells.

FIG. 16 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon the hematologicparameters: white blood cell (WBC) counts, neutrophil counts and asplatelet counts. No appreciable decrease in mean neutrophil, mean whiteblood cell or mean platelet counts was observed.

FIG. 17 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hemoglobin andreticulocyte indices. There is a minimal decrease in mean hemoglobinconcentrations at the higher dose level. In parallel there is a slightincrease in mean reticulocytosis indices

FIG. 18 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hepatic markersincluding serum aspartate transaminase (AST) and serum alaninetransaminase (ALT) along with serum albumin. There was no appreciableincreases in liver transaminases mean AST or mean ALT levels and therewas no observed change in mean albumin levels.

FIG. 19 presents the relative tumor volume of immunodeficient femaleNu/J mice (6-8 weeks old) inoculated with NCI-H292 (Non-Small Cell LungCancer) cells and administered control, pemetrexed, and Liposomal aG6intravenously at 167 mg/kg once every three weeks. As can be seen fromthese preliminary data, liposomal aG6 provides reduced tumor controlcompared to pemetrexed.

FIG. 20 presents the results of a liposomal pemetrexed alpha-Lhexaglutamate (Liposomal aG6) treatment survival study in a NSCLC (H292)xenograft model. The survival curve of mice (10) dosed intravenouslywith 90 mg/kg Liposomal aG6 once a week for four weeks (90 mg/kg subQweekly for 6 weeks) is depicted with a solid circle. The survival curveof mice (10) dosed with pemetrexed (167 mg/kg intravenously every threeweeks for 6 weeks) is depicted with a solid triangle. The dose of 167mg/kg of pemetrexed in mice is equivalent to a dose of 500 mg/m² inhumans. The survival curve of control mice (10) is depicted with an opendiamond.

FIGS. 21A-F present the dose response relationship of liposomalpemetrexed alpha-L triglutamate (Liposomal aG3), liposomal pemetrexedalpha-L pentaglutamate (Liposomal aG5), liposomal pemetrexed alpha-Loctaglutamate (Liposomal aG7), and a combination of liposomal pemetrexedalpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aG12)(Liposomal aG6 and aG12) over 48 hours on H2342 (NSCLC, adenocarcinomasubtype) (FIG. 21A), H292 (NSCLC, adenocarcinoma subtype) (FIG. 21B),HT-29 (colon cancer) (FIG. 21C), HCC1806 (triple negative breast cancer)(FIG. 21D), MCF7 (ER+ breast cancer) (FIG. 21E), and OAW28 (ovariancancer) (FIG. 21F). Cell viability was determined by CellTiter-Glo®(CTG) luminescent cell viability assay essentially as described inExample 1. As shown in all cell lines, the potency of each of thepolyglutamated pemetrexed liposomal compositions well exceeded that ofthe liposomal vehicle and empty liposome controls.

DETAILED DESCRIPTION

The disclosure generally relates to novel alpha polyglutamatedAntifolate compositions. The compositions provide advances over priortreatments of hyperproliferative diseases such as cancer. Methods ofmaking, delivering and using the alpha polyglutamated Antifolatecompositions are also provided. The alpha polyglutamated compositionshave uses that include but are not limited to treating or preventinghyperproliferative diseases such as cancer, disorders of the immunesystem including inflammation and autoimmune disease such as rheumatoidarthritis, and infectious diseases such as HIV, malaria, andschistomiasis.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

It is understood that wherever embodiments, are described herein withthe language “comprising” otherwise analogous embodiments, described interms of “containing” “consisting of” and/or “consisting essentially of”are also provided. However, when used in the claims as transitionalphrases, each should be interpreted separately and in the appropriatelegal and factual context (e.g., in claims, the transitional phrase“comprising” is considered more of an open-ended phrase while“consisting of” is more exclusive and “consisting essentially of”achieves a middle ground).

As used herein, the singular form “a”, “an”, and “the”, includes pluralreferences unless it is expressly stated or is unambiguously clear fromthe context that such is not intended.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

Headings and subheadings are used for convenience and/or formalcompliance only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. Features described under one heading or onesubheading of the subject disclosure may be combined, in variousembodiments, with features described under other headings orsubheadings. Further it is not necessarily the case that all featuresunder a single heading or a single subheading are used together inembodiments.

Unless indicated otherwise, the terms “Antifolate” and “ANTIFOL” areused interchangeably to include a salt, acid and and/or free base formof an antifolate (e.g., Antifolate disodium). Compositions containing aANTIFOL salt may further contain any of a variety of cations, such asNa+, Mg2+, K+, NH4+, and/or Ca2+. In particular embodiments, the saltsare pharmaceutically acceptable salts. In additional particularembodiments, the Antifolate salt contains Na+. Antifolates typicallycontain one L-gamma glutamyl group, and are therefore considered to bemonoglutamated for the purpose of this disclosure.

Although the compounds of the present invention can exist as a mixtureof stereoisomers it is preferred that they are resolved into oneoptically active isomeric form. Such a requirement complicates thesynthesis of the compounds and it is preferred therefore that theycontain as few asymmetric carbon atoms as possible consistent withachieving the desired activity.

As indicated previously, however, the cyclopenta[g]quinazolines of thepresent invention contain at least three asymmetric carbon atoms. Ofthese, that at the 6 position of the ring system preferably has the 6Sorientation rather than the 6R orientation. The preferred compounds (I)described hereinbefore thus preferably have such a configuration at thisasymmetric carbon atoms or less preferably are a mixture in which one orboth of these asymmetric carbon atoms is unresolved.

The antifolate can be any known or future derived folate or Antifolatethat is polyglutamated. In some embodiments, the Antifolate is selectedfrom LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolate);5-CH3-THF, 5-methyltetrahydrofolate; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX,2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT;2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-Ndelta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, Nalpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, Nalpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dPteOrn, Nalpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoicacid; 5-dH4PteOro, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,N10-propargyl-5,8-dideazafolic acid; ICI-198,583,2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583; 7-CH3-ICI-198,583,7-methyl-ICI-198,583; ZD1694,N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamicacid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,(S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaricacid; LY231514,N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamicacid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaaisofolicacid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid;N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337,3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline; andAG377,2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or astereoisomer thereof.

In some embodiments, the Antifolate is a member selected from:Aminopterin, methotrexate, raltitrexed (also referred to as TOMUDEX,ZD1694 (RTX)), plevitrexed (also referred to as BGC 9331; ZD9331),pemetrexed (also referred to as ALIMTA, LY231514), lometrexol (LMX)(5,10-dideazatetrahydrofolic acid), a cyclopenta[g]quinazoline with adipeptide ligand, CB3717, CB300945 (also referred to as BGC945) or astereoisomer thereof such as 6-R,S-BGC945 (ONX-0801), CB300638 (alsoreferred to as BGC638), and BW1843U89

The terms “polyglutamate”, polyglutamated”, or variations thereof, referto a composition comprising at least one chain of 2 or more linkedglutamyl groups. Polyglutamate chains can be linear or branched. Linearpolyglutamate chains can contain for example, glutamyl groups containingeither an alpha carboxyl group or a gamma carboxyl group linkage.Branched polyglutamate chains can comprise for example, one or moreglutamyl groups that contain both an alpha carboxyl group and a gammacarboxyl group linkage to other glutamyl groups, thereby providing abranch point of the polyglutamate. Exemplary branched polyglutamates aredepicted in FIGS. 1O-1R. Polyglutamate chains comprise an N-terminalglutamyl group and one or more C-terminal glutamyl groups. TheN-terminal glutamyl group of a polyglutamate chain is not linked toanother glutamyl group via its amine group, but is linked to one or moreglutamyl group via its carboxylic acid group. In some embodiments, theN-terminal glutamyl group of a polyglutamated-Antifolate is the glutamylgroup of the Antifolate. The C-terminal glutamyl group or groups of apolyglutamate chain are linked to another glutamyl group via their aminegroup, but are not linked to another glutamyl group via their carboxylicacid group.

The terms “polyglutamated-Antifolate”, “polyglutamated-ANTIFOL”,“ANTIFOL-PG”, “PANTIFOL” and iterations thereof, are usedinterchangeably herein to refer to a Antifolate composition thatcomprises at least one glutamyl group in addition to the glutamyl groupin the Antifolate (i.e., ANTIFOL-PGn, wherein n≥1). Reference to thenumber of glutamyl groups in a αPANTIFOL (ANTIFOL-PG) herein takes intoaccount the glutamyl group in the Antifolate. For example, an ANTIFOL-PGcomposition containing 5 glutamyl residues in addition to the glutamylgroup of ANTIFOL is referred to herein as hexaglutamated Antifolate orAntifolate hexaglutamate.

The terms “alpha glutamyl group”, “alpha glutamate”, and “alpha linkage”as they relate to the linkage of a glutamyl group, refers to a glutamylgroup that contains an alpha carboxyl group linkage. In someembodiments, the alpha linkage is an amide bond between the alphacarboxyl group of one glutamyl group and a second glutamyl group. Thealpha linkage can be between a glutamyl group and the glutamyl group inthe Antifolate, or between the glutamyl group and a second glutamylgroup that is not present in Antifolate, such as a glutamyl group withina polyglutamate chain attached to Antifolate.

The terms “gamma glutamyl group”, “gamma glutamate”, and “gammalinkage”, as they relate to the linkage of a glutamyl group, refers to aglutamyl group that contains a gamma carboxyl group linkage. Asdiscussed herein, once Antifolate enters the cell, it is polyglutamatedby the enzyme folylpoly-gamma-glutamate synthetase (FPGS), which adds Lglutamyl groups serially to the glutamyl group within the Antifolate.Consequently, alpha polyglutamated Antifolate compositions are notformed within cells during Antifolate therapy. In some embodiments, thegamma linkage is an amide bond between the gamma carboxyl group of oneglutamyl group and a second glutamyl group. The gamma linkage can bebetween a glutamyl group and the glutamyl group in the Antifolate, orbetween the glutamyl group and a second glutamyl group that is notpresent in Antifolate, such as a glutamyl group within a polyglutamatechain attached to Antifolate. In some embodiments, the gamma linkagerefers to the amide bond of the glutamyl group of the Antifolate.Reference to gamma linkages are inclusive of gamma linkage of theglutamyl group of the Antifolate unless it is expressly stated or isunambiguously clear from the context that such is not intended.

Unless indicated otherwise, the terms “alpha polyglutamated Antifolate”,αPANTIFOL”, “alpha-ANTIFOL-PG”, and iterations thereof, are usedinterchangeably herein to refer to a polyglutamated-Antifolatecomposition that comprises at least one glutamyl group that contains analpha linkage. For example, a pentaglutamated-ANTIFOL compositionwherein the 3rd glutamyl group have an alpha linkage, but each of theother glutamyl groups has a gamma linkage, is considered to be analpha-ANTIFOL-PG for the purposes of this disclosure. In someembodiments, each of the glutamyl groups of the ANTIFOL-PG other thanthe glutamyl group of ANTIFOL, have an alpha linkage (e.g., ANTIFOL-PGn,wherein n=5 and wherein each of G1, G2, G3, G4, and G5, have an alphalinkage). In some embodiments, each of the glutamyl groups of theANTIFOL-PG other than the C-terminal glutamyl group or groups and theglutamyl group of the Antifolate, has an alpha linkage (e.g.,ANTIFOL-PG_(n), wherein n=5 and wherein each of G₁, G₂, G₃, and G₄, havean alpha linkage). In some embodiments, each of the glutamyl groups ofthe PMX-PG other than the C-terminal glutamyl group or groups, has analpha linkage (e.g., ANTIFOL-PG_(n), wherein n=5 and wherein each of theglutamyl group of the Antifolate and G₁, G₂, G₃, and G₄, have an alphalinkage).

As use herein, the term “isolated” refers to a composition which is in aform not found in nature. Isolated alpha polyglutamated compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an alpha polyglutamated Antifolate which is isolated is substantiallypure. Isolated compositions will be free or substantially free ofmaterial with which they are naturally associated such as other cellularcomponents such as proteins and nucleic acids with which they maypotentially be found in nature, or the environment in which they areprepared (e.g., cell culture). The alpha polyglutamated compositions maybe formulated with diluents or adjuvants and still for practicalpurposes be isolated—for example, the alpha polyglutamated compositionswill normally be mixed with pharmaceutically acceptable carriers ordiluents when used in diagnosis or therapy. In some embodiments, theisolated alpha polyglutamated compositions (e.g., alpha polyglutamatesand delivery vehicles such as liposomes containing the alphapolyglutamate contain less than 1% or less than 0.1% undesired DNA orprotein content. In some embodiments, the alpha polyglutamatecompositions (e.g., alpha polyglutamate and delivery vehicles such asliposomes containing the alpha polyglutamate) are “isolated.”

The term “targeting moiety” is used herein to refer to a molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. The targeting moiety can comprisea wide variety of entities. Targeting moieties can include naturallyoccurring molecules, or recombinant or synthetic molecules. In someembodiments, the targeting moiety is an antibody, antigen-bindingantibody fragment, bispecific antibody or other antibody-based moleculeor compound. In some embodiments, the targeting moiety is an aptamer,avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidinbinding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, acomponent of a microorganism, a hormone, a receptor ligand or anyderivative thereof. Other targeting moieties are known in the art andare encompassed by the disclosure.

The terms “specific affinity” or “specifically binds” mean that atargeting moiety such as an antibody or antigen binding antibodyfragment, reacts or associates more frequently, more rapidly, withgreater duration, with greater affinity, or with some combination of theabove to the epitope, protein, or target molecule than with alternativesubstances, including proteins unrelated to the target epitope. Becauseof the sequence identity between homologous proteins in differentspecies, specific affinity can, in several embodiments, include abinding agent that recognizes a protein or target in more than onespecies. Likewise, because of homology within certain regions ofpolypeptide sequences of different proteins, the term “specificaffinity” or “specifically binds” can include a binding agent thatrecognizes more than one protein or target. It is understood that, incertain embodiments, a targeting moiety that specifically binds a firsttarget may or may not specifically bind a second target. As such,“specific affinity” does not necessarily require (although it caninclude) exclusive binding, e.g., binding to a single target. Thus, atargeting moiety may, in certain embodiments, specifically bind morethan one target. In certain embodiments, multiple targets may be boundby the same targeting moiety.

The term “epitope” refers to that portion of an antigen capable of beingrecognized and specifically bound by a targeting moiety (i.e., bindingmoiety) such as an antibody. When the antigen is a polypeptide, epitopescan be formed both from contiguous amino acids and noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation.

Expressions like “binding affinity for a target”, “binding to a target”and analogous expressions known in the art refer to a property of atargeting moiety which may be directly measured through thedetermination of the affinity constants, e.g., the amount of targetingmoiety that associates and dissociates at a given antigen concentration.Different methods can be used to characterize the molecular interaction,such as, but not limited to, competition analysis, equilibrium analysisand microcalorimetric analysis, and real-time interaction analysis basedon surface plasmon resonance interaction (for example using a BIACORE®instrument). These methods are well-known to the skilled person and aredescribed, for example, in Neri et al., Tibtech 14:465-470 (1996), andJansson et al., J. Biol. Chem. 272:8189-8197 (1997).

The term “delivery vehicle” refers generally to any compositions thatacts to assist, promote or facilitate entry of alpha polyglutamatedAntifolate into a cell. Such delivery vehicles are known in the art andinclude, but are not limited to, liposomes, lipospheres, polymers (e.g.,polymer-conjugates), peptides, proteins such as antibodies (e.g.,immunoconjugates, such as Antibody Drug Conjugates (ADCs)) and antigenbinding antibody fragments and derivatives thereof), cellularcomponents, cyclic oligosaccharides (e.g., cyclodextrins), micelles,microparticles (e.g., microspheres), nanoparticles (e.g., lipidnanoparticles, biodegradable nanoparticles, and core-shellnanoparticles), hydrogels, lipoprotein particles, viral sequences, viralmaterial, or lipid or liposome formulations, and combinations thereof.The delivery vehicle can be linked directly or indirectly to a targetingmoiety. In some examples, the targeting moiety is selected from among amacromolecule, a protein, a peptide, a monoclonal antibody or a fattyacid lipid.

A “subject” refers to a human or vertebrate mammal including but notlimited to a dog, cat, horse, goat and primate, e.g., monkey. Thus, theinvention can also be used to treat diseases or conditions in non-humansubjects. For instance, cancer is one of the leading causes of death incompanion animals (i.e., cats and dogs). In some embodiments, of theinvention, the subject is a human. In this disclosure, the term“subject” and “patient” is used interchangeably and has the samemeaning. It is preferred generally that a maximum dose be used, that is,the highest safe dose according to sound medical judgment.

As used herein an “effective amount” refers to a dosage of an agentsufficient to provide a medically desirable result. The effective amountwill vary with the desired outcome, the particular condition beingtreated or prevented, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent or combination therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose. In the case of cancer, the effectiveamount of an agent may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, duration ofprogression free survival (PFS), the response rates (RR), duration ofresponse, and/or quality of life.

The terms “hyperproliferative disorder”, “proliferative disease”, and“proliferative disorder”, are used interchangeably herein to pertain toan unwanted or uncontrolled cellular proliferation of excessive orabnormal cells which is undesired, such as, neoplastic or hyperplasticgrowth, whether in vitro or in vivo. In some embodiments, theproliferative disease is cancer or tumor disease (including benign orcancerous) and/or any metastases, wherever the cancer, tumor and/or themetastasis is located. In some embodiments, the proliferative disease isa benign or malignant tumor. In some embodiments, the proliferativedisease is a non-cancerous disease. In some embodiments, theproliferative disease is a hyperproliferative condition such ashyperplasias, fibrosis (especially pulmonary, but also other types offibrosis, such as renal fibrosis), angiogenesis, psoriasis,atherosclerosis and smooth muscle proliferation in the blood vessels,such as stenosis or restenosis following angioplasty.

“Cancer,” “tumor,” or “malignancy” are used as synonymous terms andrefer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (metastasize) as well as any of a number ofcharacteristic structural and/or molecular features. “Tumor,” as usedherein refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “cancerous tumor,” or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. A cancer that can be treatedusing an αPANTIFOL composition provided herein includes withoutlimitation, a non-hematologic malignancy including such as for example,lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, and melanoma; and a hematologic malignancy such as for example,a leukemia, a lymphoma and other B cell malignancies, myeloma and otherplasma cell dysplasias or dyscrasias. In some embodiments, the cancer isselected from: breast cancer, advanced head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis), bladdercancer, and central nervous system (CNS) cancer. In some embodiments,the cancer is lung cancer (e.g., NSCLC or mesothelioma). In someembodiments, the cancer is breast cancer (e.g., HER2++ or triplenegative breast cancer). In some embodiments, the cancer is colorectalcancer. In some embodiments, the cancer is ovarian cancer. In someembodiments, the cancer is endometrial cancer. In some embodiments, thecancer is pancreatic cancer. In some embodiments, the cancer is livercancer. In some embodiments, the cancer is head and neck cancer. In someembodiments, the cancer is osteosarcoma. Other types of cancer andtumors that may be treated using a αPANTIFOL composition are describedherein or otherwise known in the art. The term “metastasis” refers tospread or dissemination of a tumor, cancer or neoplasia to other sites,locations, regions or organ or tissue systems within the subject, inwhich the sites, locations regions or organ or tissue systems aredistinct from the primary tumor, cancer or neoplasia. The terms“cancer,” “cancerous,” “cell proliferative disorder,” “proliferativedisorder,” and “tumor” are not mutually exclusive as referred to herein.

Terms such as “treating,” or “treatment,” or “to treat” refer to both(a) therapeutic measures that cure, slow down, lessen symptoms of,and/or halt progression of a diagnosed pathologic condition or disorderand (b) prophylactic or preventative measures that prevent and/or slowthe development of a targeted disease or condition. Thus, subjects inneed of treatment include those already with the cancer, disorder ordisease; those at risk of having the cancer or condition; and those inwhom the infection or condition is to be prevented. Subjects areidentified as “having or at risk of having” cancer, an infectiousdisease, a disorder of the immune system, a hyperproliferative disease,or another disease or disorder referred to herein using well-knownmedical and diagnostic techniques. In certain embodiments, a subject issuccessfully “treated” according to the methods provided herein if thesubject shows, e.g., total, partial, or transient amelioration orelimination of a symptom associated with the disease or condition (e.g.,cancer, rheumatoid arthritis). In specific embodiments, the termstreating,” or “treatment,” or “to treat” refer to the amelioration of atleast one measurable physical parameter of a proliferative disorder,such as growth of a tumor, not necessarily discernible by the patient.In other embodiments, the terms treating,” or “treatment,” or “to treat”refer to the inhibition of the progression of a proliferative disorder,either physically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments, the terms treating,” or “treatment,” or “totreat” refer to the reduction or stabilization of tumor size, tumor cellproliferation or survival, or cancerous cell count. Treatment can bewith a α-PANTIFOL composition, alone or in combination with anadditional therapeutic agent.

“Subject” and “patient,” and “animal” are used interchangeably and referto mammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, rats, and mice, and other animals.Animals include all vertebrates, e.g., mammals and non-mammals, such aschickens, amphibians, and reptiles. “Mammal” as used herein refers toany member of the class Mammalia, including, without limitation, humansand nonhuman primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs, and other members of theclass Mammalia known in the art. In a particular embodiment, the patientis a human.

“Treatment of a proliferative disorder” is used herein to includemaintaining or decreasing tumor size, inducing tumor regression (eitherpartial or complete), inhibiting tumor growth, and/or increasing thelife span of a subject having the proliferative disorder. In oneembodiment, the proliferative disorder is a solid tumor. Such tumorsinclude, for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma. In one embodiment, theproliferative disorder is a hematologic malignancy. Such hematologicmalignancies include for example, a leukemia, a lymphoma and other Bcell malignancies, myeloma and other plasma cell dysplasias ordyscrasias. In some embodiments, the cancer is selected from: breastcancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma,Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL),mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, andchorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralnervous system (CNS) cancer. In some embodiments, the cancer is lungcancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer isbreast cancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the cancer is colorectal cancer. In some embodiments, thecancer is ovarian cancer. In some embodiments, the cancer is endometrialcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

The terms “inflammation” and “inflammatory disease” are usedinterchangeably and refer to a disease or disorder characterized orcaused by inflammation. “Inflammation” refers to a local response tocellular injury that is marked by capillary dilatation, leukocyticinfiltration, redness, heat, and pain that serves as a mechanisminitiating the elimination of noxious agents and of damaged tissue. Thesite of inflammation includes the lungs, the pleura, a tendon, a lymphnode or gland, the uvula, the vagina, the brain, the spinal cord, nasaland pharyngeal mucous membranes, a muscle, the skin, bone or bonytissue, a joint, the urinary bladder, the retina, the cervix of theuterus, the canthus, the intestinal tract, the vertebrae, the rectum,the anus, a bursa, a follicle, and the like. Such inflammatory diseasesinclude, but are not limited to, inflammatory bowel disease, rheumatoiddiseases (e.g., rheumatoid arthritis), other arthritic diseases (e.g.,acute arthritis, acute gouty arthritis, bacterial arthritis, chronicinflammatory arthritis, degenerative arthritis (osteoarthritis),infectious arthritis, juvenile arthritis, mycotic arthritis, neuropathicarthritis, polyarthritis, proliferative arthritis, psoriatic arthritis,venereal arthritis, viral arthritis), fibrositis, pelvic inflammatorydisease, acne, psoriasis, actinomycosis, dysentery, biliary cirrhosis,Lyme disease, heat rash, Stevens-Johnson syndrome, mumps, pemphigusvulgaris, and blastomycosis. Inflammatory bowel diseases are chronicinflammatory diseases of the gastrointestinal tract which include,without limitation, Crohn's disease, ulcerative colitis, andindeterminate colitis. Rheumatoid arthritis is a chronic inflammatorydisease primarily of the joints, usually polyarticular, marked byinflammatory changes in the synovial membranes and articular structuresand by muscle atrophy and rarefaction of the bones.

The term “therapeutic agent” is used herein to refer to an agent or aderivative or prodrug thereof, that can interact with ahyperproliferative cell such as a cancer cell or an immune cell, therebyreducing the proliferative status of the cell and/or killing the cell.Examples of therapeutic agents include, but are not limited to,chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g.,cisplatin, carboplatin, oxaliplatin), taxanes (e.g., TAXOL®), etoposide,alkylating agents (e.g., cyclophosphamide, ifosamide), metabolicantagonists (e.g., Antifolate (ANTIFOL), 5-fluorouracil gemcitabine, orderivatives thereof), antitumor antibiotics (e.g., mitomycin,doxorubicin), plant-derived antitumor agents (e.g., vincristine,vindesine, TAXOL®). Such agents may further include, but are not limitedto, the anticancer agent trimetrexate, temozolomide, raltitrexed,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG),bis-chloronitrosourea (BCNU) and CAMPTOTHECIN™, or a therapeuticderivative of any thereof. Additional examples of therapeutic agentsthat may be suitable for use in accordance with the disclosed methodsinclude, without limitation, anti-restenosis, pro- oranti-proliferative, anti-inflammatory, anti-neoplastic, antimitotic,anti-platelet, anticoagulant, antifibrin, antithrombin, cytostatic,antibiotic and other anti-infective agents, anti-enzymatic,anti-metabolic, angiogenic, cytoprotective, angiotensin convertingenzyme (ACE) inhibiting, angiotensin II receptor antagonizing and/orcardioprotective agents. “Therapeutic agents” also refer to salts,acids, and free based forms of the above agents.

As used herein, the term “chemotherapeutic agent” when used in relationto cancer therapy, refers to any agent that results in the death ofcancer cells or inhibits the growth or spread of cancer cells. Examplesof such chemotherapeutic agents include alkylating agents, antibiotics,antimetabolitic agents, plant-derived agents, and hormones. In someembodiments, the chemotherapeutic agent is cisplatin. In someembodiments, the chemotherapeutic agent is carboplatin. In someembodiments, the chemotherapeutic agent is oxaliplatin. In otherembodiments, the chemotherapeutic agent is gemcitabine. In otherembodiments, the chemotherapeutic agent is doxorubicin.

The term “antimetabolite” is used herein to refer to a therapeutic agentthat inhibits the utilization of a metabolite or a prodrug thereof.Examples of antimetabolites include Antifolate, pemetrexed,5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine,5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs suchas nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine,azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, andcladribine. Anti-metabolites useful for practicing the disclosed methodsinclude nucleoside analogs, including a purine or pyrimidine analogs. Insome embodiments, the alpha polyglutamated Antifolate compositions areused in combination with an antimetabolite selection fromfluoropyrimidine 5-fluorouracil, 5-fluoro-2′-deoxycytidine, cytarabine,gemcitabine, troxacitabine, decitabine, Azacytidine, pseudoisocytidine,Zebularine, Ancitabine, Fazarabine, 6-azacytidine, capecitabine,N4-octadecyl-cytarabine, elaidic acid cytarabine, fludarabine,cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or aderivative thereof. In one example, the nucleoside analog is a substratefor a nucleoside deaminase that is adenosine deaminase or cytidinedeaminase. In some examples, the nucleoside analog is selected fromamong fludarabine, cytarabine, gemcitabine, decitabine and azacytidineor derivatives thereof. In certain embodiments, the antimetabolite is5-fluorouracil.

As used herein, a “taxane” is an anti-cancer agent that interferes withor disrupts microtubule stability, formation and/or function. Taxaneagents include paclitaxel and docetaxel as well as derivatives thereof,wherein the derivatives function against microtubules by the same modeof action as the taxane from which they are derived. In certainembodiments, the taxane is paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the taxane is paclitaxel (TAXOL®),docetaxel (TAXOTERE®), albumin-bound paclitaxel (nab-paclitaxel;ABRAXANE®), DHA-paclitaxel, or PG-paclitaxel.

The term “pharmaceutically-acceptable carrier” A “pharmaceuticallyacceptable carrier” refers to an ingredient in a pharmaceuticalformulation, other than an active ingredient, which is nontoxic to asubject. A pharmaceutically acceptable carrier includes, but is notlimited to, a buffer, excipient, stabilizer, or preservative.Pharmaceutically-acceptable carriers can include for example, one ormore compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration to a human or othersubject.

This disclosure generally relates novel alpha polyglutamated Antifolate(ANTIFOL) compositions and methods of making and using the compositionsto treat diseases including hyperproliferative diseases such as cancer,disorders of the immune system such as rheumatoid arthritis, andinfectious diseases such as HIV, malaria, and schistomiasis.

In some embodiments, the disclosure provides:

-   [1] A composition comprising an alpha polyglutamated Antifolate,    wherein at least one glutamyl group has an alpha carboxyl group    linkage;-   [2] the composition of [1], wherein the Antifolate is selected from:    piritrexim, pralatrexate, AG2034, GW1843, and LY309887, or a    stereoisomer thereof;-   [3] the composition of [1], wherein the Antifolate is selected from:    PMX, MTX, RTX, and LMX, or a stereoisomer thereof;-   [4] the composition according to [1], wherein the Antifolate is    selected from: LV (etoposide), L-leucovorin    (L-5-formyltetrahydrofolate); 5-CH3-THF, 5-methyltetrahydrofolate;    FA, folic acid; PteGlu, pteroyl glutamate (FA); MTX, methotrexate;    2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT,    Antifolate; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT,    2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin;    PT523, N alpha-(4-amino-4-deoxypteroyl)-N    delta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),    5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,    5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,    N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, N    alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N    alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid;    5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;    5-dH4PteAPBA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phospho-butanoic    acid; 5-dH4PteOro, N    alpha-(5-deaza-5,6,7,8-tetrahydropt-eroyl)-L-ornithine; CB3717,    N10-propargyl-5,8-dideazafolic acid; ICI-198,583,    2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;    4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,    4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;    Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583;    7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694,    N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic    acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,    (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaric    acid; LY231514,    N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic    acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;    2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu,    5-deazaaisofolic acid; N9-CH3-5-d(i)PteGlu,    N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu,    N9-formyl-5-deazaisofolic acid; AG337,    3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline;    and 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)    amino]quinazoline; or a stereoisomer thereof;-   [5] the composition of [1], wherein the Antifolate is selected from:    methotrexate, raltitrexed, plevitrexed, pemetrexed, lometrexol (LMX;    5,10-dideazatetrahydrofolic acid), a cyclopenta[g]quinazoline with a    dipeptide ligand, CB3717, CB300945, or a stereoisomer thereof, such    as 6-R,S-BGC 945 (ONX-0801), CB300638, and BW1843U89;-   [6] the composition according to any of [1]-[5], wherein,    -   (a) each of the glutamyl groups of the polyglutamated Antifolate        other than the glutamyl group of the Antifolate has an alpha        carboxyl group linkage; or    -   (b) two or more glutamyl groups of the polyglutamated Antifolate        have a gamma carboxyl group linkage;-   [7] the composition according to any of [1]-[5], wherein,    -   (a) each of the glutamyl groups other than the C-terminal        glutamyl group or groups and the glutamyl group of the        Antifolate has an alpha carboxyl group linkage; or    -   (b) each of the glutamyl groups other than the C-terminal        glutamyl group or groups has an alpha carboxyl group linkage;-   [8] the composition according to any of [1]-[7], wherein the alpha    polyglutamated Antifolate:    -   (a) contains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl        groups;    -   (b) is an alpha pentaglutamated Antifolate; or    -   (c) is an alpha hexaglutamated Antifolate;-   [9] the composition according to any of [1]-[8], wherein the alpha    polyglutamated Antifolate comprises 1-10 glutamyl groups having an    alpha carboxyl group linkage;-   [10] the composition according to any of [1]-[9], wherein:    -   (a) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form,    -   (b) each of the glutamyl groups of the alpha polyglutamated        Antifolate is in the L-form,    -   (c) at least 1 of the glutamyl groups of the alpha        polyglutamated Antifolate is in the D-form,    -   (d) each of the glutamyl groups of the alpha polyglutamated        Antifolate other than the glutamyl group of the Antifolate is in        the D-form, or    -   (e) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form and at least 1 of        the glutamyl groups is in the D-form;-   [11] the composition according to any of [1]-[10], wherein the    polyglutamate is linear;-   [12] the composition according to any of [1]-[10], wherein the    polyglutamate is branched;-   [13] a liposomal composition comprising the alpha polyglutamated    Antifolate according to any of [1]-[12] (Lp-αPANTIFOL);-   [14] the Lp-αPANTIFOL composition of [13], wherein the alpha    polyglutamated Antifolate is selected from:    -   (a) AG2034, piritrexim, pralatrexate, GW1843, Antifolate, and        LY309887; or    -   (b) PMX, MTX, RTX, and LMX, or a stereoisomer thereof;-   [15] the Lp-αPANTIFOL composition of [13], wherein the    polyglutamated Antifolate is selected from: LV (etoposide),    L-leucovorin (L-5-formyltetrahydrofolate); 5-CH3-THF,    5-methyltetrahydrofolate; FA, folic acid; PteGlu, pteroyl glutamate    (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX,    2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT;    2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,    10-ethyl-10-deazaaminopterin; PT523, N    alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine;    DDATHF (lometrexol), 5,10-dideaza-5,6,7,8-tetrahydrofolic acid;    5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroisofolic acid;    N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic    acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid;    5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic    acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine;    5-dH4PteHCysA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;    5-dH4PteAPBA, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoic    acid; 5-dH4PteOro, N    alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,    N10-propargyl-5,8-dideazafolic acid; ICI-198,583,    2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;    4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,    4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;    Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583;    7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694,    N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic    acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,    (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaric    acid; LY231514,    N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic    acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;    2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu,    5-deazaaisofolic acid; N9-CH3-5-d(i)PteGlu,    N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu,    N9-formyl-5-deazaisofolic acid; AG337,    3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline;    and AG377,    2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline;    or a stereoisomer thereof;-   [16] the Lp-αPANTIFOL composition according to [13], wherein the    Antifolate is selected from: methotrexate, raltitrexed, plevitrexed,    pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), a    cyclopenta[g]quinazoline with a dipeptide ligand, CB3717, CB300945,    or a stereoisomer thereof, such as 6-R,S-BGC 945 (ONX-0801),    CB300638, and BW1843U89;-   [17] the Lp-αPANTIFOL composition according to any of [13]-[16],    wherein the liposome comprises an alpha polyglutamated Antifolate    containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups;-   [18] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha tetraglutamated Antifolate;-   [19] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha pentaglutamated Antifolate;-   [20] the Lp-αPANTIFOL composition according to any of [13]-[17],    wherein the liposome comprises an alpha hexaglutamated Antifolate;-   [21] the Lp-αPANTIFOL composition according to any of [13]-[20],    wherein the polyglutamate is linear or branched;-   [22] the Lp-αPANTIFOL composition according to any of [13]-[21],    wherein:    -   (a) each of the glutamyl groups other than the glutamyl group of        the Antifolate has an alpha carboxyl group linkage, or    -   (b) two or more glutamyl groups have a gamma carboxyl group        linkage;-   [23] the Lp-αPANTIFOL composition according to any of [13]-[21],    wherein:    -   (a) each of the glutamyl groups other than the C-terminal        glutamyl group or groups and the glutamyl group of the        Antifolate has an alpha carboxyl group linkage, or    -   (b) each of the glutamyl groups other than the C-terminal        glutamyl group or groups has an alpha carboxyl group linkage;-   [24] the Lp-αPANTIFOL composition according to any of [13]-[23],    wherein    -   (a) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form,    -   (b) each of the glutamyl groups of the alpha polyglutamated        Antifolate is in the L-form,    -   (c) at least 1 of the glutamyl groups of the alpha        polyglutamated Antifolate is in the D-form,    -   (d) each of the glutamyl groups of the alpha polyglutamated        Antifolate other than the glutamyl group of the Antifolate is in        the D-form, or    -   (e) at least 2 of the glutamyl groups of the alpha        polyglutamated Antifolate are in the L-form and at least 1 of        the glutamyl groups is in the D-form;-   [25] the Lp-αPANTIFOL composition according to any of [13]-[24],    wherein the liposome is pegylated (PαLp-αPANTIFOL);-   [26] the Lp-αPANTIFOL composition according to any of [13]-[24],    wherein the liposome is not pegylated;-   [27] the Lp-αPANTIFOL composition according to any of [13]-[26],    wherein the liposome has a diameter in the range of 20 nm to 200 nm;-   [28] the Lp-αPANTIFOL composition according to any of [13]-[27],    wherein the polyglutamate is linear or branched;-   [29] the Lp-αPANTIFOL composition according to any of [13]-[28],    wherein the liposomes comprise at least 1% weight by weight (w/w) of    the alpha polyglutamated Antifolate or wherein during the process of    preparing the Lp-αPANTIFOL, at least 1% of the starting material of    alpha polyglutamated Antifolate is encapsulated (entrapped) in the    Lp-αPANTIFOL;-   [30] the Lp-αPANTIFOL composition according to any of [13]-[29],    wherein the liposome has a diameter in the range of 20 nm to 500 nm    or 20 nm to 200 nm;-   [31] the Lp-αPANTIFOL composition according to any of [13]-[29],    wherein the liposome has a diameter in the range of 80 nm to 120 nm;-   [32] the Lp-αPANTIFOL composition according to any of [13]-[31],    wherein the liposome is formed from liposomal components;-   [33] the Lp-αPANTIFOL composition according to [32], wherein the    liposomal components comprise at least one of an anionic lipid and a    neutral lipid;-   [34] the Lp-αPANTIFOL composition according to [32] or [33], wherein    the liposomal components comprise at least one selected from: DSPE;    DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol;    cholesterol-PEG; and cholesterol-maleimide;-   [35] the Lp-αPANTIFOL composition according to any of [32]-[34],    wherein the liposomal components comprise at least one selected    from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide;    cholesterol; and HSPC;-   [36] the Lp-αPANTIFOL composition according to any of [32]-[35],    wherein one or more liposomal components further comprises a steric    stabilizer;-   [37] the Lp-αPANTIFOL composition according to [36], wherein the    steric stabilizer is at least one selected from polyethylene glycol    (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl    pyrrolidone) (PVP); poly(acrylamide) (PAA);    poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl    polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic    poly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol,    copolymer containing polyethylene glycol and polypropylene oxide,    Poloxamer 188, and polyvinyl alcohol;-   [38] the Lp-αPANTIFOL composition according to [37], wherein the    steric stabilizer is PEG and the PEG has a number average molecular    weight (Mn) of 200 to 5000 daltons;-   [39] the Lp-αPANTIFOL composition according to any of [13]-[38],    wherein the liposome is anionic or neutral;-   [40] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is less than or equal    to zero;-   [41] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is between 0 to −150    mV;-   [42] the Lp-αPANTIFOL composition according to any of [13]-[39],    wherein the liposome has a zeta potential that is between −30 to −50    mV;-   [43] the Lp-αPANTIFOL composition according to any of [13]-[38],    wherein the liposome is cationic;-   [44] the Lp-αPANTIFOL composition according to any of [13]-[43],    wherein the liposome has an interior space comprising the alpha    polyglutamated Antifolate and an aqueous pharmaceutically acceptable    carrier;-   [45] the Lp-αPANTIFOL composition of [44], wherein the    pharmaceutically acceptable carrier comprises a tonicity agent such    as dextrose, mannitol, glycerine, potassium chloride, sodium    chloride, at a concentration of greater than 1%;-   [46] the Lp-αPANTIFOL composition of [44], wherein the aqueous    pharmaceutically acceptable carrier is trehalose;-   [47] the Lp-αPANTIFOL composition of [46], wherein the    pharmaceutically acceptable carrier comprises 5% to 20% weight of    trehalose;-   [48] the Lp-αPANTIFOL composition according to any of [44]-[47],    wherein the pharmaceutically acceptable carrier comprises 1% to 15    weight of dextrose;-   [49] the Lp-αPANTIFOL composition according to any of [44]-[48],    wherein the interior space of the liposome comprises 5% dextrose    suspended in an HEPES buffered solution;-   [50] the Lp-αPANTIFOL composition according to any of [44]-[49],    wherein the pharmaceutically acceptable carrier comprises a buffer    such as HEPES Buffered Saline (HBS) or similar, at a concentration    of between 1 to 200 mM and a pH of between 2 to 8;-   [51] the Lp-αPANTIFOL composition according to any of [44]-[50],    wherein the pharmaceutically acceptable carrier comprises a total    concentration of sodium acetate and calcium acetate of between 50 mM    to 500 mM;-   [52] the Lp-αPANTIFOL composition according to any of [13]-[51],    wherein the interior space of the liposome has a pH of 5-8 or a pH    of 6-7, or any range therein between;-   [53] the Lp-αPANTIFOL composition according to any of [13]-[52],    wherein the liposome comprises less than 500,000 or less than    200,000 molecules of the alpha polyglutamated Antifolate;-   [54] the Lp-αPANTIFOL composition according to any of [13]-[53],    wherein the liposome comprises between 10 to 100,000 molecules of    the alpha polyglutamated Antifolate, or any range therein between;-   [55] the Lp-αPANTIFOL composition according to any of [13]-[54],    which further comprises a targeting moiety and wherein the targeting    moiety has a specific affinity for a surface antigen on a target    cell of interest;-   [56] the Lp-αPANTIFOL composition according to [55], wherein the    targeting moiety is attached to one or both of a PEG and the    exterior of the liposome, optionally wherein targeting moiety is    attached to one or both of the PEG and the exterior of the liposome    by a covalent bond;-   [57] the Lp-αPANTIFOL composition of [55] or [56], wherein the    targeting moiety is a polypeptide;-   [58] the Lp-αPANTIFOL composition according to any of [55]-[57],    wherein the targeting moiety is an antibody or an antigen binding    fragment of an antibody;-   [59] the Lp-αPANTIFOL composition according to any of [55]-[58],    wherein the targeting moiety binds the surface antigen with an    equilibrium dissociation constant (Kd) in a range of 0.5×10-10 to    10×10-6 as determined using BIACORE® analysis;-   [60] the Lp-αPANTIFOL composition according to any of [55]-[59],    wherein the targeting moiety specifically binds one or more folate    receptors selected from: folate receptor alpha (FR-α), folate    receptor beta (FR-β), and folate receptor delta (FR-δ);-   [61] the Lp-αPANTIFOL composition according to any of [55]-[60],    wherein the targeting moiety comprises one or more selected from: an    antibody, a humanized antibody, an antigen binding fragment of an    antibody, a single chain antibody, a single-domain antibody, a    bi-specific antibody, a synthetic antibody, a pegylated antibody,    and a multimeric antibody;-   [62] the Lp-αPANTIFOL composition according to any of [55]-[61],    wherein each pegylated liposome comprises from 1 to 1000 or 30-200    targeting moieties;-   [63] the Lp-αPANTIFOL composition according to any of [44]-[57],    further comprising one or more of an immunostimulatory agent, a    detectable marker and a maleimide, wherein the immunostimulatory    agent, the detectable marker or the maleimide is attached to said    PEG or the exterior of the liposome;-   [64] the Lp-αPANTIFOL composition of [63], wherein the    immunostimulating agent is at least one selected from: a protein    immunostimulating agent; a nucleic acid immunostimulating agent; a    chemical immunostimulating agent; a hapten; and an adjuvant;-   [65] the Lp-αPANTIFOL composition of [63] or [64], wherein the    immunostimulating agent is at least one selected from: a    fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta    glucan; a beta-1,3-glucan; a beta-1,6-glucan; a resolvin (e.g., a    Resolvin D such as Dn-6DPA or Dn-3DPA, a Resolvin E. or a T series    resolvin); and a Toll-like receptor (TLR) modulating agent such as,    an oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and an    eritoran lipid (e.g., E5564);-   [66] the Lp-αPANTIFOL composition according to any of [63]-[65],    wherein the immunostimulatory agent and the detectable marker is the    same;-   [67] the Lp-αPANTIFOL composition according to any of [63]-[66],    further comprising a hapten;-   [68] the Lp-αPANTIFOL composition of [67], wherein the hapten    comprises one or more of fluorescein or Beta 1, 6-glucan;-   [69] The Lp-αPANTIFOL composition according to any of [13]-[68],    which further comprises at least one cryoprotectant selected from    mannitol; trehalose; sorbitol; and sucrose;-   [70] a targeted composition comprising the composition according to    any of [1]-[69];-   [71] an non-targeted composition comprising the composition    according to any of [1]-[54] and [64]-[69];-   [72] the Lp-αPANTIFOL composition according to any of [13]-[71],    which further comprises carboplatin and/or pembroluzumab;-   [73] a pharmaceutical composition comprising the liposomal alpha    polyglutamated Antifolate composition according to any of [13]-[72];-   [74] a pharmaceutical composition comprising alpha polyglutamated    Antifolate composition according to any of [1]-[8];-   [75] the composition of any of [1]-[74], for use in the treatment of    disease;-   [76] use of the composition of any of [1]-[75], in the manufacture    of a medicament for the treatment of disease;-   [77] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the composition of any of [1]-[75] to the subject;-   [78] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74] to the subject;-   [79] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the composition of any of    [1]-[74];-   [80] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the liposomal alpha    polyglutamated Antifolate composition of any of [13]-[74];-   [81] the method of [79] or [80], wherein the hyperproliferative cell    is a cancer cell, a mammalian cell, and/or a human cell;-   [82] a method for treating cancer that comprises administering an    effective amount of the composition of any of [1]-[74] to a subject    having or at risk of having cancer;-   [83] a method for treating cancer that comprises administering an    effective amount of the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[73] to a subject having or at risk of    having cancer;-   [84] the method of [82] or [83], wherein the cancer is selected    from: a non-hematologic malignancy including such as for example,    lung cancer, pancreatic cancer, breast cancer, ovarian cancer,    prostate cancer, head and neck cancer, gastric cancer,    gastrointestinal cancer, colorectal cancer, esophageal cancer,    cervical cancer, liver cancer, kidney cancer, biliary duct cancer,    gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma),    brain cancer, central nervous system cancer, and melanoma; and a    hematologic malignancy such as for example, a leukemia, a lymphoma    and other B cell malignancies, myeloma and other plasma cell    dyscrasias.-   [85] the method of [82] or [83], wherein the cancer is selected    from: the cancer is a member selected from: breast cancer, advanced    head and neck cancer, lung cancer, stomach cancer, osteosarcoma,    Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL),    mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,    chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma    (desmoid tumors, aggressive fibromatosis), bladder cancer, and    Central Nervous System (CNS) lymphoma;-   [86] the method of [82] or [83], wherein the cancer is selected    from: colorectal cancer, lung cancer, breast cancer, head and neck    cancer, and pancreatic cancer;-   [87] the method of [82] or [83], wherein the cancer is a sarcoma    such as osteosarcoma;-   [88] a method for treating cancer that comprises administering an    effective amount of the Lp-αPANTIFOL composition of any of [55]-[71]    to a subject having or at risk of having a cancer cell that    expresses on its surface a folate receptor bound by the targeting    moiety;-   [89] a maintenance therapy comprising administering an effective    amount of the composition of any of [1]-[74] to a subject that is    undergoing or has undergone cancer therapy;-   [90] a maintenance therapy comprising administering an effective    amount of the liposomal alpha polyglutamated Antifolate composition    of any of [13]-[74] to a subject that is undergoing or has undergone    cancer therapy;-   [91] a method for treating a disorder of the immune system that    comprises administering an effective amount of the composition of    any of [1]-[74] to a subject having or at risk of having a disorder    of the immune system, optionally wherein the disorder of the immune    system is selected from: inflammation (e.g., acute and chronic),    systemic inflammation, rheumatoid arthritis, inflammatory bowel    disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic    lupus erythematosus, and Takayasu, and psoriasis;-   [92] a method for treating a disorder of the immune system that    comprises administering an effective amount of the liposomal alpha    polyglutamated Antifolate composition of any of [9]-[74] to a    subject having or at risk of having a disorder of the immune system,    optionally wherein the disorder of the immune system is selected    from: inflammation (e.g., acute and chronic), systemic inflammation,    rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn    disease, dermatomyositis/polymyositis, systemic lupus erythematosus,    and Takayasu, and psoriasis;-   [93] a method for treating:    -   (a) an infectious disease that comprises administering an        effective amount of the composition according to any of [1]-[74]        to a subject having or at risk of having an infectious disease;    -   (b) an infectious disease, cardiovascular disease, metabolic        disease, or another disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having an        infectious disease, cardiovascular disease, or another disease,        wherein the disease is a member selected from: atherosclerosis,        cardiovascular disease (CVD), coronary artery disease,        myocardial infarction, stroke, metabolic syndrome, a gestational        trophoblastic disease, and ectopic pregnancy;    -   (c) an autoimmune disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having an        autoimmune disease;    -   (d) rheumatoid arthritis, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having rheumatoid        arthritis;    -   (e) an inflammatory condition that comprises administering an        effective amount of the composition according to of any of any        of [1]-[74] to a subject having or at risk of having        inflammation, optionally wherein the inflammation is acute,        chronic, and/or systemic inflammation; or    -   (f) a skin condition that comprises administering an effective        amount of the composition according to of any of claims any of        [1]-[74] to a subject having or at risk of having a skin        condition, optionally wherein the skin condition is psoriasis;-   [94] a method for treating an infectious disease that comprises    administering an effective amount of the liposomal alpha    polyglutamated Antifolate composition of any of [13]-[74] to a    subject having or at risk of having an infectious disease;-   [95] a method of delivering alpha polyglutamated Antifolate to a    tumor expressing a folate receptor on its surface, the method    comprising: administering the Lp-αPANTIFOL composition of any of    [1]-[74] to a subject having the tumor in an amount to deliver a    therapeutically effective dose of the alpha polyglutamated    Antifolate to the tumor;-   [96] a method of preparing an alpha polyglutamated Antifolate    composition comprising the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74], the method comprising: forming a    mixture comprising: liposomal components and alpha polyglutamated    antifolate in solution; homogenizing the mixture to form liposomes    in the solution; and processing the mixture to form liposomes    containing alpha polyglutamated Antifolate;-   [97] a method of preparing an alpha polyglutamated Antifolate    composition comprising the liposomal alpha polyglutamated Antifolate    composition of any of [13]-[74], the method comprising: forming a    mixture comprising: liposomal components and alpha polyglutamated    Antifolate in solution; and processing the mixture to form liposomes    containing alpha polyglutamated Antifolate;-   [98] the method of [97], wherein the processing the mixture    comprises homogenizing the mixture to form liposomes in the    solution;-   [99] a method of preparing the composition of any of [55]-[74]    comprising the steps of: forming a mixture comprising: liposomal    components and alpha polyglutamated Antifolate in a solution;    homogenizing the mixture to form liposomes in the solution;    processing the mixture to form liposomes entrapping and/or    encapsulating alpha polyglutamated Antifolate; and providing a    targeting moiety on a surface of the liposomes, the targeting moiety    having specific affinity for at least one of folate receptor alpha    (FR-α), folate receptor beta (FR-β) and folate receptor delta    (FR-δ);-   [100] a method of preparing the composition of any of [55]-[74],    comprising the steps of: forming a mixture comprising: liposomal    components and alpha polyglutamated Antifolate in a solution;    processing the mixture to form liposomes entrapping and/or    encapsulating alpha polyglutamated Antifolate; and providing a    targeting moiety on a surface of the liposomes, the targeting moiety    having specific affinity for at least one of folate receptor alpha    (FR-α), folate receptor beta (FR-β) and folate receptor delta    (FR-δ);-   [101] the method of [100], wherein the processing step comprises    homogenizing the mixture to form liposomes in the solution;-   [102] the method according to any of [99] to [101], wherein the    processing step includes one or more steps of: thin film hydration,    extrusion, in-line mixing, ethanol injection technique,    freezing-and-thawing technique, reverse-phase evaporation, dynamic    high pressure microfluidization, microfluidic mixing, double    emulsion, freeze-dried double emulsion, 3D printing, membrane    contactor method, and stirring; and-   [103] the method according to any of [99] to [102], wherein said    processing step includes one or more steps of modifying the size of    the liposomes by one or more of steps of extrusion, high-pressure    microfluidization, and/or sonication;-   [104] the method of any of [96] to [103], wherein at least 1% of the    starting material of alpha polyglutamated Antifolate is encapsulated    or entrapped in the Lp-αPANTIFOL.

II. Alpha Polyglutamated Antifolate (αPANTIFOL)

The disclosure generally relates alpha polyglutamated Antifolate(αPANTIFOL) compositions. The αPANTIFOL compositions comprise at leastone glutamyl group having an alpha linkage. These compositions arestructurally distinct from the L-gamma polyglutamated forms ofAntifolate (LαPANTIFOL) that are produced by the enzymefolylpoly-gamma-glutamate synthetase (FPGS) in cells during Antifolatetherapy.

In some embodiments, the αPANTIFOL composition contains 2-20, 2-15,2-10, 2-5, or more than 5, glutamyl groups (including the glutamyl groupof the Antifolate). In some embodiments, each of the glutamyl groups inthe αPANTIFOL other than the glutamyl group of the Antifolate, has analpha linkage. In some embodiments, each of the glutamyl groups in theαPANTIFOL other than the C-terminal glutamyl group or groups and theglutamyl group of the Antifolate, has an alpha linkage. In someembodiments, each of the glutamyl groups in the αPANTIFOL other than theC-terminal glutamyl group or groups has an alpha linkage. In someembodiments, 2 or more of the glutamyl groups in the αPANTIFOL have agamma linkage. In some embodiments, at least one glutamyl group of thealpha polyglutamated Antifolate has both an alpha carboxyl group linkageand a gamma carboxyl group linkage. In some embodiments, each of theglutamyl groups in the αPANTIFOL is in the L-form. In some embodiments,each of the glutamyl groups in the αPANTIFOL other than the glutamylgroup of the Antifolate, is in the D-form. In some embodiments, theαPANTIFOL comprises two or more glutamyl groups in the L-form and one ormore glutamyl groups in the D-form. In some embodiments, thepolyglutamate chain of the αPANTIFOL is linear (not branched). In someembodiments, the polyglutamate chain of the αPANTIFOL is branched.

In some embodiments, the Antifolate is selected from: PMX, MTX, RTX, andLMX, or a stereoisomer thereof.

In some embodiments, the Antifolate is selected from: LV (etoposide),L-leucovorin (L-5-formyltetrahydrofolate); 5-CH3-THF,5-methyltetrahydrofolate; FA, folic acid; PteGlu, pteroyl glutamate(FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX,2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT;2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-Ndelta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, Nalpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, Nalpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dPteOrn, Nalpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoicacid; 5-dH4PteOro, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,N10-propargyl-5,8-dideazafolic acid; ICI-198,583,2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583; 7-CH3-ICI-198,583,7-methyl-ICI-198,583; ZD1694,N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamicacid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,(S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaricacid; LY231514,N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamicacid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaaisofolicacid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid;N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337,3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline; andAG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or a stereoisomer thereof.

In some embodiments, the Antifolate is selected from: methotrexate,raltitrexed, plevitrexed, pemetrexed, lometrexol (LMX;5,10-dideazatetrahydrofolic acid), a cyclopenta[g]quinazoline with adipeptide ligand, CB3717, CB300945, or a stereoisomer thereof, such as6-R,S-BGC 945 (ONX-0801), CB300638, and BW1843U89.

In some embodiments, the Antifolate is a 6-substitutedpyrrolo[2,3-d]pyrimidine benzoyl antifolate. In some embodiments, theAntifolate is a 6-substituted pyrrolo[2,3-d]pyrimidine benzoylantifolate with carbon bridge length from 1- to 6-carbons (e.g., acompound having the structure of Formula (I) A below, wherein n1=1-6).In some embodiments, the Antifolate is a 6-substitutedthieno[2,3-d]pyrimidine benzoyl antifolates with bridge with a bridgelength from 2-8 carbons (e.g., a compound having the structure ofFormula (II), wherein n2=7-13). In some embodiments, the Antifolate is a6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoylreplacement for the benzoyl moiety with a bridge length from 2-8 carbons(e.g., a compound having the structure of Formula (III), whereinn1=1-6). In some embodiments, the Antifolate has a structure accordingto any of Formula (I)-(III), wherein x=4, 5, 6, 2-10, 4-6, or more than5.

In some embodiments, the Antifolate is selected from: an indoline ringand modified ornithine-bearing methotrexate derivative, an indoline ringand modified glutamic acid-bearing methotrexate derivative, analkyl-substituted benzene ring C bearing methotrexate derivative, abenzoxazine moiety-bearing methotrexate derivative, a benzothiazinemoiety-bearing methotrexate derivative, a 10-deazaminopterin analog, a5-deazaminopterin methotrexate analog, a 5,10-dideazaminopterinmethotrexate analog, a indoline moiety-bearing methotrexate derivative,a lipophilic amide methotrexate derivative, aL-threo-(2S,4S)-4-fluoro-glutamic acid containing methotrexate analog, aDL-3,3-difluoroglutamic acid-containing methotrexate analog, amethotrexate tetrahydroquinazoline analog, a N-(ac-aminoacyl)methotrexate derivative, a biotin methotrexate derivative, a D-glutamicacid methotrexate analog, a D-erythrou, threo-4-fluoroglutamic acidmethotrexate analog, β,γ-methano methotrexate analog, a10-deazaminopterin (10-EDAM) analog, a γ-tetrazole methotrexate analog,a N-(L-α-aminoacyl) methotrexate derivative, a meta isomer ofaminopterin, an ortho isomer of aminopterin, ahydroxymethylmethotrexate, a γ-fluoromethotrexate, a polyglutamylmethotrexate derivative, a gem-diphosphonate methotrexate analog (see,e.g., WO1988/06158, the contents of which is herein incorporated byreference in its entirety), a α-substituted methotrexate analog, aγ-substituted methotrexate analog, a 5-methyl-5-deaza methotrexateanalog (see. e.g., U.S. Pat. No. 4,725,687, the contents of each ofwhich is herein incorporated by reference in its entirety), an Ndelta-acyl-N α-(4-amino-4-deoxypteroyl)-L-ornithine derivative, a8-deaza methotrexate analog, an acivicin methotrexate analog, apolymeric platinol methotrexate derivative, amethotrexate-γ-dimyristoylphophatidylethanolamine, a methotrexatepolyglutamate analog, a poly-γ-glutamyl methotrexate derivative, adeoxyuridylate methotrexate derivative, a iodoacetyl lysine methotrexateanalog, a 2,omega.-diaminoalkanoid acid-containing methotrexate analog,a polyglutamate methotrexate derivative, a 5-methyl-5-deaza analog, aquinazoline methotrexate analog, a pyrazine methotrexate analog, acysteic or homocysteic acid methotrexate analog (see, e.g., U.S. Pat.No. 4,490,529, and EPA 0142220, the contents of each of which is hereinincorporated by reference in its entirety), a 7-tert-butyl methotrexateester, a fluorinated methotrexate analog, a folate methotrexate analog,a phosphonoglutamic acid analog, a poly (L-lysine) methotrexateconjugate, a dilysine or trilysine methotrexate derivate, a7-hydroxymethotrexate, a poly-γ-glutamyl methotrexate analog, a3′,5′-dichloromethotrexate, a diazoketone or chloromethylketonemethotrexate analog, a 10-propargylaminopterin, an alkyl methotrexatehomologs, a lectin derivative of methotrexate, a polyglutamatemethotrexate derivative, a halogenated methotrexate derivative, a8-alkyl-7,8-dihydro analog, a 7-methyl methotrexate derivative, adichloromethotrexate, a lipophilic methotrexate derivative, a3′,5′-dichloromethotrexate, a deaza amethopterin analog, and MX068; or astereoisomer thereof.

In some embodiments, the Antifolate has the Formula (IV):

wherein X=CH₂, C₂H₄, or O(CH₂)₃O; R1=Me or Et; R2=H, Cl, F, OH, orR2=R3; and R3=H, Cl, F, OH, Me, or Br.

In some embodiments, the Antifolate has the Formula (IV) wherein, X=CH2;R1=Me or Et; R2=H, Cl, F, OH, or R2=R3; and R3=H, Cl, F, OH, Me, or Br.In some embodiments, X=CH₂; R1=Me; R2=H, Cl, F, OH; and R3=H, Cl, Me, orBr. In some embodiments, X=O(CH₂)₃O; R1=Me; and R2=R3=H.

In some embodiments, the Antifolate has the Formula (V):

wherein X=C₂H₄, C₄H₈, C₆H₁₂, O(CH2)₂O, or O(CH₂)₃O; R1=H or Cl, orR2=R3; and R3=H or Cl.

In some embodiments, the Antifolate has the Formula (V) wherein, X=C₂H₄;and R1=R2=H or CL. In some embodiments, X=C₂H₄; R1=Cl; and R2=H. In someembodiments, X=C₄H₈; and R1=R2=H. In some embodiments, X=C₆H₁₂; andR1=R2=H.

In some embodiments, the Antifolate has the Formula (VI):

wherein X=CH₂ or C₂H₄; Y=2,5-thiophene; and R=CH₂F, Cn, Et, Me, orCH₂OH.

In some embodiments, the Antifolate has the Formula (VI) wherein, X=CH₂;Y=2,5-thiophene; and R=H₂F, Cn, Et, or CH₂OH. In some embodiments,X=C₂H₄; Y=2,5-thiophene; and R=Me.

In some embodiments, the Antifolate has the Formula (VII):

wherein X=N or CH; Y=NH₂; CH₃, or H; and R=CH₃, CHO, or H.

In some embodiments, the Antifolate has the Formula (VII) wherein, (a)X=N; Y=NH₂; and R=H; (b) X=N; Y=NH₂; and R=CH₃; (c) X=N, Y=NH₂; andR=CHO; (d) X=CH, Y=NH₂, R=H; (e) X=CH, Y=H, R=H; or (f) X=CH, Y=CH₃, andR=H.

In some embodiments, the Antifolate has the Formula (VIII):

wherein A=NH, NCH₃, or CH₂.

In some embodiments, the Antifolate has the Formula (IX):

wherein, (a) X=OH; R=H; and Y=GIu, (b) X=OCH₃; R=H; and Y=GIu, (c) X=OH;R=H; and Y=Valine; (d) X=OH; R=H; and Y=Suberate; or (e) X=OH; R=CH₃;and Y=GIu.

In additional embodiments, the Antifolate is a cyclopenta[g]quinazolinederivative. In some embodiments, the cyclopenta[g]quinazoline derivativeisN—{N-{4-[N-(2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; orN—{N-{4-[N-(2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, the Antifolate has the Formula (X):

wherein R1 is H, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 hydroxyalkyl orC1-4 fluoroalkyl;

-   R2 is hydrogen, C1-4 alkyl, C3-4 alkenyl, C3-4 alkynyl, C2-4    hydroxyalkyl C2-4 halogenoalkyl or C1-4 cyanoalkyl;-   Ar is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or    pyrimidinediyl which may optionally bear one or two substituents    selected from halogeno, hydroxy, amino, nitro, cyano,    trifluoromethyl, C1-4 alkyl and C1-4 alkoxy; and-   R3 is a group of one of the following formulae:    —NHCH(CO2H)-A1-Y1-NH-A3-Y3 or R3 is an alpha or gamma carboxyl    linked L- or D-glutamyl group.

In some embodiments, the Antifolate has the Formula (X) wherein, R1 isC1-4 alkyl or C1-4 hydroxyalkyl (e.g., a methyl or a hydroxymethyl); R2is (a) methyl, ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxy-ethyl,2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl, (b) methyl or (c)prop-2-ynyl; and Ar is 1,4-phenylene or a 1,4-phenylene having one ortwo substituents selected from chloro and fluoro (e.g. a 2-fluorosubstituent such as 2-fluoro-1,4-phenylene or2,6-difluoro-1,4-phenylene), thiophene-2,5-diyl, thiazole-2,5-diyl orpyridine-2,5-diyl.

In some embodiments, the Antifolate has the Formula (X) wherein, R1 ismethyl or hydroxymethyl; R2 is methyl or prop-2-ynyl; and Ar is1,4-phenylene or 1,4-phenylene having a 2-fluoro substituent as in2,6-difluoro-1,4-phenylene or especially 2-fluoro-1,4-phenylene or ispyridine 2,5-diyl. In some embodiments, Ar is 1,4-phenylene or2-fluoro-1,4-phenylene.

In other embodiments, the alpha polyglutamated Antifolate is acyclopenta[g]quinazoline disclosed in WO2009/115776, WO 2003/020300, WO2003/020706, WO 2003/020748, Gibbs et al., Cancer Research 65 (15):11721-11728 (2005), and Bavetsias et al., Tetrahedron 63(7):1537-1543(2007), the contents of each of which is herein incorporated byreference in its entirety.

In some embodiments, the alpha polyglutamated Antifolate isdiglutamated. That is, the alpha polyglutamated Antifolate contains 1additional glutamyl group in addition to the glutamyl group in theAntifolate (αANTIFOL-PG1), and the additional glutamyl group is linkedto the glutamyl group in the Antifolate through an alpha linkage. Insome embodiments, each of the glutamyl groups of the alpha diglutamatedAntifolate is in the L-form. In other embodiments, the alphadiglutamated Antifolate comprises a glutamyl group in the D-form.

In some embodiments, the alpha polyglutamated Antifolate istriglutamated. That is, the alpha polyglutamated Antifolate contains 2additional glutamyl groups in addition to the glutamyl group in theAntifolate (αANTIFOL-PG2). In some embodiments, each of the 2 additionalglutamyl groups have an alpha linkage. In other embodiments, one of the2 additional glutamyl groups have an alpha linkage and the otherglutamyl group has a gamma linkage. In some embodiments, one of the 2additional glutamyl groups has an alpha linkage. In some embodiments,one of the 2 additional glutamyl groups has a gamma linkage. In someembodiments, two of the three glutamyl groups have an alpha linkage. Inother embodiments, one of the three glutamyl groups has an alpha linkageand another glutamyl group has a gamma linkage. In some embodiments, oneglutamyl group has both an alpha linkage and a gamma linkage. In someembodiments, each of the glutamyl groups of the alpha triglutamatedAntifolate is in the L-form. In other embodiments, the alphatriglutamated Antifolate comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the alphatriglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thetriglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate istetraglutamated and thus contains 3 additional glutamyl groups inaddition to the glutamyl group of the Antifolate (αANTIFOL-PG3). In someembodiments, each of the 3 additional glutamyl groups have an alphalinkage. In other embodiments, 1 or 2 of the 3 additional glutamylgroups have an alpha linkage and the remaining 2 or 1 glutamyl groups,respectively, have a gamma linkage. In some embodiments, 2 of the 3additional glutamyl groups have an alpha linkage. In other embodiments,one of the 3 additional glutamyl groups has an alpha linkage and anotheradditional glutamyl group has a gamma linkage. In other embodiments, oneof the 3 additional glutamyl groups has an alpha linkage and a gammalinkage. In other embodiments, three of the four glutamyl groups have analpha linkage. In some embodiments, at least one glutamyl group has bothan alpha linkage and a gamma linkage. In some embodiments, the alphatetraglutamated Antifolate comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphatetraglutamated Antifolate is in the L-form. In other embodiments, thealpha tetraglutamated Antifolate comprises a glutamyl group in theD-form. In further embodiments, each of the glutamyl groups of the alphatetraglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thetetraglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate ispentaglutamated (αANTIFOL-PG4) and contains a chain of 4 additionalglutamyl groups attached to the glutamyl group in the Antifolate. Insome embodiments, each of the 4 additional glutamyl groups in the chainhave an alpha linkage. In some embodiments, each of the 4 additionalglutamyl groups in the chain other than the C-terminal glutamyl group orgroups have an alpha linkage. In other embodiments, 1, 2, or 3, of the 4additional glutamyl groups have an alpha linkage and the remaining 3, 2,or 1, glutamyl groups, respectively, are linked to a glutamyl group ofthe molecule through a gamma linkage. In other embodiments, 1 or 2 ofthe 4 additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 5 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 5glutamyl groups in the chain other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, the alphapentaglutamated Antifolate comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphapentaglutamated Antifolate is in the L-form. In other embodiments, thealpha pentaglutamated Antifolate comprises a glutamyl group in theD-form. In further embodiments, each of the glutamyl groups of the alphapentaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thepentaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate ishexaglutamated (αANTIFOL-PG5) and contains a chain of 5 additionalglutamyl groups attached to the glutamyl group in the Antifolate. Insome embodiments, each of the 5 additional glutamyl groups in the chainhave an alpha linkage. In some embodiments, each of the 5 additionalglutamyl groups in the chain other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 4 of the 5 additionalglutamyl groups in the chain have an alpha linkage. In otherembodiments, 1, 2, 3, or 4, of the 5 additional glutamyl groups arelinked to a glutamyl group of the molecule through an alpha linkage andthe remaining 4, 3, 2, or 1, glutamyl groups, respectively, are linkedto a glutamyl group of the molecule through a gamma linkage. In otherembodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups have analpha linkage and the remaining non-C-terminal glutamyl groups arelinked to a glutamyl group of the molecule through a gamma linkage. Insome embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 6 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 6 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 5 of the 6 glutamyl groups have an alpha linkage. In someembodiments, the alpha hexaglutamated Antifolate comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the alpha hexaglutamated Antifolate is in the L-form.In other embodiments, the alpha hexaglutamated Antifolate comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the alpha hexaglutamated Antifolate other than theglutamyl group of the Antifolate, is in the D-form. In additionalembodiments, the hexaglutamated Antifolate comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate isheptaglutamated (αANTIFOL-PG6) and thus contains a chain of 6 additionalglutamyl groups attached to the glutamyl group in the Antifolate. Insome embodiments, each of the 6 additional glutamyl groups have an alphalinkage. In some embodiments, each of the 6 additional glutamyl groupsin the chain other than the C-terminal glutamyl group or groups have analpha linkage. In some embodiments, 5 of the 6 additional glutamylgroups in the chain have an alpha linkage. In other embodiments, 1, 2,3, 4, or 5, of the 6 additional glutamyl groups have an alpha linkageand the remaining 5, 4, 3, 2, or 1, glutamyl groups, respectively, havea gamma linkage. In other embodiments, 1, 2, 3, 4, or 5 of the 6additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 7 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 7glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 6 of the 7 glutamyl groups havean alpha linkage. In some embodiments, the alpha heptaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. Infurther embodiments, each of the glutamyl groups of the alphaheptaglutamated Antifolate is in the L-form. In other embodiments, thealpha heptaglutamated Antifolate comprises a glutamyl group in theD-form. In further embodiments, each of the glutamyl groups of the alphaheptaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, theheptaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isoctaglutamated (αANTIFOL-PG7) and thus contains a chain of 7 additionalglutamyl groups attached to the glutamyl group in the Antifolate. Insome embodiments, each of the 7 additional glutamyl groups in the chainother than the C-terminal glutamyl group or groups have an alphalinkage. In some embodiments, 6 of the 7 additional glutamyl groups inthe chain have an alpha linkage. In some embodiments, each of the 7additional glutamyl groups have an alpha linkage. In other embodiments,1, 2, 3, 4, 5, or 6, of the 7 additional glutamyl groups have an alphalinkage and the remaining 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,or 6 of the 7 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 8 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 8 glutamyl groups other than the C-terminal glutamyl group or groupshave an alpha linkage. In some embodiments, 7 of the 8 glutamyl groupshave an alpha linkage. In some embodiments, the alpha octaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. Infurther embodiments, each of the glutamyl groups of the alphaoctaglutamated Antifolate is in the L-form. In other embodiments, thealpha octaglutamated Antifolate comprises a glutamyl group in theD-form. In further embodiments, each of the glutamyl groups of the alphaoctaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, theoctaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isnonaglutamated (αANTIFOL-PG8) and contains a chain of 8 additionalglutamyl groups attached to the glutamyl group in the Antifolate. Insome embodiments, each of the 8 additional glutamyl groups in the chainother than the C-terminal glutamyl group or groups have an alphalinkage. In some embodiments, 7 of the 8 additional glutamyl groups inthe chain have an alpha linkage. In some embodiments, each of the 8additional glutamyl groups have an alpha linkage. In other embodiments,1, 2, 3, 4, 5, 6, or 7, of the 8 additional glutamyl groups have analpha linkage and the remaining 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, or 7 of the 8 additional glutamyl groups have an alpha linkage andthe remaining non-C-terminal glutamyl groups are linked to a glutamylgroup of the molecule through a gamma linkage. In some embodiments, atleast one additional glutamyl group has both an alpha linkage and agamma linkage. In some embodiments, at least one of the 9 glutamylgroups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 9 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 8of the 9 glutamyl groups have an alpha linkage. In some embodiments, thealpha nonaglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In further embodiments, each of the glutamyl groups of thealpha nonaglutamated Antifolate is in the L-form. In other embodiments,the alpha nonaglutamated Antifolate comprises a glutamyl group in theD-form. In further embodiments, each of the glutamyl groups of the alphanonaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thenonaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isdecaglutamated (αANTIFOL-PG9) (i.e., contains a chain of 9 additionalglutamyl groups attached to the glutamyl group in the Antifolate). Insome embodiments, each of the 9 additional glutamyl groups have an alphalinkage. In some embodiments, each of the 9 additional glutamyl groupsin the chain other than the C-terminal glutamyl group or groups have analpha linkage. In some embodiments, 8 of the 9 additional glutamylgroups in the chain have an alpha linkage. In other embodiments, 1, 2,3, 4, 5, 6, 7, or 8, of the 9 additional glutamyl groups have an alphalinkage and the remaining 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, or 8 of the 9 additional glutamyl groups have an alpha linkage andthe remaining non-C-terminal glutamyl groups are linked to a glutamylgroup of the molecule through a gamma linkage. In some embodiments, atleast one additional glutamyl group has both an alpha linkage and agamma linkage. In some embodiments, at least one of the 10 glutamylgroups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 10 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 9of the 10 glutamyl groups have an alpha linkage. In some embodiments,the alpha decaglutamated Antifolate comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha decaglutamated Antifolate is in the L-form. In otherembodiments, the alpha decaglutamated Antifolate comprises a glutamylgroup in the D-form. In further embodiments, each of the glutamyl groupsof the alpha decaglutamated Antifolate other than the glutamyl group ofthe Antifolate, is in the D-form. In additional embodiments, thedecaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isundecaglutamated (αANTIFOL-PG10). In some embodiments, each of the 10additional glutamyl groups have an alpha linkage. In some embodiments,each of the 10 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 9 of the 10 additional glutamyl groups in the chain have analpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9, ofthe 10 additional glutamyl groups have an alpha linkage and theremaining 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively,have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9of the 10 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 11 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 11 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 10 of the 11 glutamylgroups have an alpha linkage. In some embodiments, the alphaundecaglutamated Antifolate comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphaundecaglutamated Antifolate is in the L-form. In other embodiments, thealpha undecaglutamated Antifolate comprises a D glutamyl group. Infurther embodiments, each of the glutamyl groups of the alphaundecaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, theundecaglutamated Antifolate comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isdodecaglutamated (αANTIFOL-PG11). In some embodiments, each of the 11additional glutamyl groups have an alpha linkage. In some embodiments,each of the 11 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 10 of the 11 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, of the 11, additional glutamyl groups have an alpha linkage and theremaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 12glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 12 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 11of the 12 glutamyl groups have an alpha linkage. In some embodiments,the alpha dodecaglutamated Antifolate comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha dodecaglutamated Antifolate is in the L-form. Inother embodiments, the alpha dodecaglutamated Antifolate comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the alpha dodecaglutamated Antifolate other than theglutamyl group of the Antifolate, is in the D-form. In additionalembodiments, the dodecaglutamated Antifolate comprises a glutamyl groupin the D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate istridecaglutamated (αANTIFOL-PG12). In some embodiments, each of the 12additional glutamyl groups have an alpha linkage. In some embodiments,each of the 12 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 11 of the 12 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11, of the 12 additional glutamyl groups have an alpha linkage andthe remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 13glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 13 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 12of the 13 glutamyl groups have an alpha linkage. In some embodiments,the alpha tridecaglutamated Antifolate comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha tridecaglutamated Antifolate is in the L-form. Inother embodiments, the alpha tridecaglutamated Antifolate comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the alpha tridecaglutamated Antifolate other than theglutamyl group of the Antifolate, is in the D-form. In additionalembodiments, the tridecaglutamated Antifolate comprises a glutamyl groupin the D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate istetradecaglutamated (αANTIFOL-PG13). In some embodiments, each of the 13additional glutamyl groups have an alpha linkage. In some embodiments,each of the 13 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 12 of the 13 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12, of the 13 additional glutamyl groups have an alpha linkageand the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamylgroups, respectively, have a gamma linkage. In other embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groupshave an alpha linkage and the remaining non-C-terminal glutamyl groupsare linked to a glutamyl group of the molecule through a gamma linkage.In some embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 14 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 14 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 13 of the 14 glutamyl groups have an alpha linkage. In someembodiments, the alpha tetradecaglutamated Antifolate comprises two ormore glutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the alpha tetradecaglutamated Antifolate is in theL-form. In other embodiments, the alpha tetradecaglutamated Antifolatecomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the alpha tetradecaglutamated Antifolate otherthan the glutamyl group of the Antifolate, is in the D-form. Inadditional embodiments, the tetradecaglutamated Antifolate comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate ispentadecaglutamated (αANTIFOL-PG14). In some embodiments, each of the 14additional glutamyl groups have an alpha linkage. In some embodiments,each of the 14 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 13 of the 14 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13, of the 14 additional glutamyl groups have an alphalinkage and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1,glutamyl groups, respectively, have a gamma linkage. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 15 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 15glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 14 of the 15 glutamyl groups havean alpha linkage. In some embodiments, the alpha pentadecaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. Infurther embodiments, each of the glutamyl groups of the alphapentadecaglutamated Antifolate is in the L-form. In other embodiments,the alpha pentadecaglutamated Antifolate comprises a glutamyl group inthe D-form. In further embodiments, each of the glutamyl groups of thealpha pentadecaglutamated Antifolate other than the glutamyl group ofthe Antifolate, is in the D-form. In additional embodiments, thepentadecaglutamated Antifolate comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate ishexadecaglutamated (αANTIFOL-PG15). In some embodiments, each of the 15additional glutamyl groups have an alpha linkage. In some embodiments,each of the 15 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 14 of the 15 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14, of the 15 additional glutamyl groups have an alphalinkage and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1, glutamyl groups, respectively, have a gamma linkage. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 16 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 16glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 15 of the 16 glutamyl groups havean alpha linkage. In some embodiments, the alpha hexadecaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. Infurther embodiments, each of the glutamyl groups of the alphahexadecaglutamated Antifolate is in the L-form. In other embodiments,the alpha hexadecaglutamated Antifolate comprises a glutamyl group inthe D-form. In further embodiments, each of the glutamyl groups of thealpha hexadecaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thehexadecaglutamated Antifolate comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In other embodiments, the alpha polyglutamated Antifolate isheptadecaglutamated (αANTIFOL-PG16). In some embodiments, each of the 16additional glutamyl groups have an alpha linkage. In some embodiments,each of the 16 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 15 of the 16 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15, of the 16, additional glutamyl groups have analpha linkage and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage. Inother embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15of the 16 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 17 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 17 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 16 of the 17 glutamylgroups have an alpha linkage. In some embodiments, the alphaheptadecaglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In further embodiments, each of the glutamyl groups of thealpha heptadecaglutamated Antifolate is in the L-form. In otherembodiments, the alpha heptadecaglutamated Antifolate comprises a Dglutamyl group. In further embodiments, each of the glutamyl groups ofthe alpha heptadecaglutamated Antifolate other than the glutamyl groupof the Antifolate, is in the D-form. In additional embodiments, theheptadecaglutamated Antifolate comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isoctadecaglutamated (αANTIFOL-PG17). In some embodiments, each of the 17additional glutamyl groups have an alpha linkage. In some embodiments,each of the 17 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 16 of the 17 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or 16, of the 17 additional glutamyl groups have analpha linkage and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 of the 17 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 18 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 18 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 17 of the 18 glutamylgroups have an alpha linkage. In some embodiments, the alphaoctadecaglutamated Antifolate comprises two or more glutamyl groups inthe L-form. In further embodiments, each of the glutamyl groups of thealpha octadecaglutamated Antifolate is in the L-form. In otherembodiments, the alpha octadecaglutamated Antifolate comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the alpha octadecaglutamated Antifolate other thanthe glutamyl group of the Antifolate, is in the D-form. In additionalembodiments, the octadecaglutamated Antifolate comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form. Insome embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate isnonadecaglutamated (αANTIFOL-PG18). In some embodiments, each of the 18additional glutamyl groups have an alpha linkage. In some embodiments,each of the 18 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 17 of the 18 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, or 17, of the 18 additional glutamyl groups havean alpha linkage and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gammalinkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 19glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 19 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 18of the 19 glutamyl groups have an alpha linkage. In some embodiments,the alpha nonadecaglutamated Antifolate comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha nonadecaglutamated Antifolate is in the L-form. Inother embodiments, the alpha nonadecaglutamated Antifolate comprises a Dglutamyl group. In further embodiments, each of the glutamyl groups ofthe alpha nonadecaglutamated Antifolate other than the glutamyl group ofthe Antifolate, is in the D-form. In additional embodiments, thenonadecaglutamated Antifolate comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate isicosaglutamated d(αANTIFOL-PG19). In some embodiments, each of the 19additional glutamyl groups have an alpha linkage. In some embodiments,each of the 19 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 18 of the 19 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18, of the 19 additional glutamyl groupshave an alpha linkage and the remaining 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have agamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups havean alpha linkage and the remaining non-C-terminal glutamyl groups arelinked to a glutamyl group of the molecule through a gamma linkage. Insome embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 20 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 20 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 19 of the 20 glutamyl groups have an alpha linkage. In someembodiments, the alpha icosaglutamated Antifolate comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the alpha icosaglutamated Antifolate is in theL-form. In other embodiments, the alpha icosaglutamated Antifolatecomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the alpha icosaglutamated Antifolate otherthan the glutamyl group of the Antifolate, is in the D-form. Inadditional embodiments, the icosaglutamated Antifolate comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate ishenicosaglutamated (αANTIFOL-PG20). In some embodiments, each of the 20additional glutamyl groups have an alpha linkage. In some embodiments,each of the 20 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 19 of the 20 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19, of the 20 additional glutamylgroups have an alpha linkage and the remaining 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 21 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 21glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 20 of the 21 glutamyl groups havean alpha linkage. In some embodiments, the alpha henicosaglutamatedAntifolate comprises two or more glutamyl groups in the L-form. Infurther embodiments, each of the glutamyl groups of the alphahenicosaglutamated Antifolate is in the L-form. In other embodiments,the alpha henicosaglutamated Antifolate comprises a glutamyl group inthe D-form. In further embodiments, each of the glutamyl groups of thealpha henicosaglutamated Antifolate other than the glutamyl group of theAntifolate, is in the D-form. In additional embodiments, thehenicosaglutamated Antifolate comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated Antifolate contains achain of 4-7 glutamyl groups attached to Antifolate (i.e., αANTIFOL-PGn,wherein n=4-7) and each of the 4-7 attached glutamyl groups have analpha linkage. In some embodiments, the alpha polyglutamated Antifolatecontains a chain of 4-7 glutamyl groups attached to the Antifolate(i.e., αANTIFOL-PGn, wherein n=4-7) and each of the 4-7 attachedglutamyl groups other than the C-terminal glutamyl group or groups hasan alpha linkage. In some embodiments, each of the 4-7 attached glutamylgroups is in the L-form. In other embodiments, each of the 4-7 attachedglutamyl groups is in the D-form. In other embodiments, the 4-7 attachedglutamyl groups are in the L-form and the D-form. In some embodiments,the polyglutamate chain is linear. In other embodiments, thepolyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate (αPANTIFOL)contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10,4-7, or 4-6, glutamyl groups including the glutamyl group of theAntifolate, or any range therein between. In some embodiments, each ofthe glutamyl groups in the αPANTIFOL other than the glutamyl group ofthe Antifolate have an alpha linkage. In some embodiments, each of theglutamyl groups in the αPANTIFOL other than the C-terminal glutamylgroup or groups and the glutamyl group of the Antifolate has an alphalinkage. In some embodiments, each of the glutamyl groups in theαPANTIFOL other than the C-terminal glutamyl group or groups has analpha linkage. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14, of the glutamyl groups in the αPANTIFOL have an alphalinkage. In some embodiments, the αPANTIFOL comprises glutamyl groups inthe L-form and the D-form. In further embodiments, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14, of the glutamyl groups in the αPANTIFOLhave an alpha linkage and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, ornone, of the glutamyl groups, respectively, has a gamma linkage. In someembodiments, each of the glutamyl groups in the polyglutamate structureof the polyglutamated Antifolate is in the L-form. In some embodiments,each of the glutamyl groups in the αPANTIFOL other than the glutamylgroup of the Antifolate is in the D-form. In one embodiment, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, of the glutamyl groups in theαPANTIFOL is in the L-form. In another embodiment, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14, of the glutamyl groups in the αPANTIFOL isin the D-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated Antifolate (αPANTIFOL)contains a total of 2-20, 2-15, 2-10, 2-5, glutamyl groups including theglutamyl group of the Antifolate, or any range therein between. In someembodiments, each of the glutamyl groups in the αPANTIFOL other than theglutamyl group of the Antifolate, have an alpha linkage. In someembodiments, each of the glutamyl groups in the αPANTIFOL other than theC-terminal glutamyl group or groups and the glutamyl group of theAntifolate has an alpha linkage. In some embodiments, each of theglutamyl groups in the αPANTIFOL other than the C-terminal glutamylgroup or groups has an alpha linkage. In other embodiments, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of theglutamyl groups have an alpha linkage. In some embodiments, theαPANTIFOL contains two or more glutamyl groups having a gamma linkage.In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, or 19, of the glutamyl groups in the αPANTIFOL otherthan the glutamyl group of the Antifolate. In some embodiments, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of theglutamyl groups have an alpha linkage. In further embodiments, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of theglutamyl groups in the αPANTIFOL other than the glutamyl group of theAntifolate have an alpha linkage and 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or none, of the glutamyl groups,respectively, have a gamma linkage. In some embodiments, each of theglutamyl groups in the αPANTIFOL is in the L-form. In some embodiments,each of the glutamyl groups in the αPANTIFOL other than the glutamylgroup of the Antifolate is in the D-form. In one embodiment, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, of theglutamyl groups in the αPANTIFOL are in the L-form. In anotherembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, or 19, glutamyl groups in the αPANTIFOL is in the D-form.

In some embodiments, the alpha polyglutamated Antifolate contains atotal of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, glutamylgroups in addition to the glutamyl group of the Antifolate). In furtherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, ofthe additional glutamyl groups have an alpha linkage. In additionalembodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, of theglutamyl groups in the alpha polyglutamated Antifolate have a gammalinkage. In some embodiments, at least one glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, the glutamylgroup in the Antifolate has an alpha linkage. In some embodiments, theglutamyl group in the Antifolate has both an alpha linkage and a gammalinkage.

In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15, glutamyl groups in the alpha polyglutamated Antifolateare in the L-form, the D-form, or in the L-form and the D-form. In someembodiments, each of the glutamyl groups of the alpha polyglutamatedAntifolate is in the L-form. In other embodiments, each of the glutamylgroups of the alpha polyglutamated Antifolate other than the glutamylgroup of the Antifolate is in the D-form. In alternative embodiments, atleast two of the glutamyl groups in the alpha polyglutamated Antifolateare in the L-form and at least one of the glutamyl groups in the alphapolyglutamated Antifolate is in the D-form. In some embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, glutamyl groups inthe alpha polyglutamated Antifolate are in the L-form. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, glutamylgroups in the alpha polyglutamated Antifolate are in the D-form.

In additional embodiments, the alpha polyglutamated Antifolate contains20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100, alphaglutamyl groups, or any range therein between. In some embodiments, eachof the glutamyl groups of the alpha polyglutamated Antifolate is in theL-form. In other embodiments, each of the glutamyl groups of the alphapolyglutamated Antifolate other than the glutamyl group of theAntifolate is in the D-form. In alternative embodiments, at least two ofthe glutamyl groups in the alpha polyglutamated Antifolate are in theL-form and at least one of the glutamyl groups in the alphapolyglutamated Antifolate is in the D-form

In additional embodiments, the provided compositions comprise an alphapolyglutamated Antifolate that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10,or 1-20, glutamyl groups that have alpha linkages. In some embodiments,the alpha polyglutamated Antifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9,1-10, or 1-20, glutamyl groups in the L-form. In some embodiments, thealpha polyglutamated Antifolate contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,1-10, or 1-20, glutamyl groups in the D-form. In some embodiments, thealpha polyglutamated Antifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9,1-10, or 1-20, glutamyl groups in the L-form and 0, 1, 2, 3, 4, 5, 6, 7,8, 9, or 1-10 or 1-20, glutamyl groups in the D-form.

In other embodiments, the alpha polyglutamated Antifolate contains atleast 1 glutamyl group that has both an alpha linkage and a gammalinkage. In some embodiments, the alpha polyglutamated Antifolatecontains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10, glutamylgroups that have both an alpha linkage and a gamma linkage.

In some embodiments, the alpha-polyglutamated Antifolate contains aleast 1 glutamyl group having an alpha linkage and contains comprises 2,3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more, glutamyl groups having a gammalinkage. For example, in some embodiments, the alpha polyglutamatedAntifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, L-alpha glutamylgroup linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10,L-gamma glutamyl group linkages. In some further embodiments, the alphapolyglutamated Antifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10,L-alpha glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6,7, 8, 9, or 1-10, D-gamma glutamyl group linkages. In additional furtherembodiments, the alpha polyglutamated Antifolate contains 1, 2, 3, 4, 5,6, 7, 8, 9, or 1-10, D-alpha glutamyl group linkages and furthercontains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-gamma glutamyl grouplinkages. In additional further embodiments, the alpha polyglutamatedAntifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-alpha glutamylgroup linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10,D-gamma glutamyl group linkages. In other further embodiments, the alphapolyglutamated Antifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10,D-gamma glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6,or 1-10, L-gamma glutamyl group linkages. In other embodiments, thealpha polyglutamated Antifolate contains at least 1 glutamyl group thathas both an alpha linkage and a gamma linkage. In some embodiments, thealpha polyglutamated Antifolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9,1-10, or more than 10, glutamyl groups that have both an alpha linkageand a gamma linkage.

In some embodiments, the alpha polyglutamated Antifolate compositionprovided herein is capable of accepting one or more additional glutamylgroups, that is the composition is able to act as a substrate for byFPGS (folylpolyglutamate synthetase). Reagents and assays and reagentsfor determining the ability of an alpha polyglutamated Antifolatecomposition to act as a substrate for FPGS (e.g., human FPGS, or ratliver FPGS) are readily available and can routinely be performed.

In some embodiments, the rate of uptake of naked alpha PANTIFOLcompositions disclosed herein (e.g., alpha PANTIFOL that is notassociated with a delivery vehicle) by hepatic cells is significantlyreduced compared to the uptake rate of the Antifolate under physiologicconditions. In some embodiments, the rate of hepatic cell uptake of thenaked alpha PANTIFOL composition is less than 30%, 20%, 15%, or 10%compared to the rate of the Antifolate. In further embodiments, the rateof the efflux (transport out) of alpha PANTIFOL compositions disclosedherein from hepatic-cells occurs at a rate that is significantly reducedcompared to the Antifolate (e.g., less than 30%, 20%, 15%, or 10%)compared to the rate of the Antifolate.

In some embodiments, an alpha polyglutamated Antifolate compositionprovided herein is more cytotoxic to hyperproliferative cells than theAntifolate. In some embodiments the hyperproliferative cells are cancercells. In some embodiments, the hyperproliferative cells a colorectalcarcinoma cells, colon cancer cells, breast cancer cells, or ovariancancer cells. In some embodiments, the cancer cells are mesotheliomacells or non-small cell lung carcinoma cells. In some embodiments,cytotoxicity is measured in an in vitro assay. In some embodiments, thealpha polyglutamated Antifolate is a hexaglutamated Antifolate.

In some embodiments, an alpha polyglutamated Antifolate compositionprovided herein has lower toxic side effects than the Antifolate. Insome embodiments, the alpha polyglutamated Antifolate compositionprovided herein is less toxic to non-hyperproliferative cells than theAntifolate. In some embodiments, the alpha polyglutamated Antifolatecomposition provided herein is less toxic to neutrophils, liver cells,or to colon epithelium cells than the Antifolate. In some embodiments,the neutrophils human neutrophils, differentiating human neutrophils, orneutrophils differentiated from CD34+ cells. In some embodiments, theliver cells are AML12 liver cells. In some embodiments, the colonepithelium cells are CCD841 colon epithelium cells. In some embodiments,the toxicity is measured in an in vitro assay. In some embodiments, thealpha polyglutamated Antifolate is a hexaglutamated Antifolate.

In some embodiments, an alpha polyglutamated Antifolate compositionprovided herein has lower toxic side effects than the Antifolate. Insome embodiments, an alpha polyglutamated Antifolate compositionprovided herein causes fewer or less severe toxic side effects in a vivoassay than the Antifolate. In some embodiments, the in vivo assay is anin vivo murine model. In some embodiments, an alpha polyglutamatedAntifolate composition provided herein causes fewer or less severehematological or hepatic toxic side effects than the Antifolate. In someembodiments, hematological side effects are assessed by measuring meanneutrophil, mean white blood cell or mean platelet counts. In someembodiments, hepatic toxic side effects are assessed by measuring serumaspartate transaminase (AST), serum alanine transaminase (ALT), and/orserum albumin levels. In some embodiments, the in vivo assay comprisesadministering 40 mg/kg or 80 mg/kg of the alpha polyglutamatedAntifolate composition once weekly for 4 weeks. In some embodiments, thealpha polyglutamated Antifolate is a hexaglutamated Antifolate.

In some embodiments, treatment with an alpha polyglutamated Antifolatecomposition provided herein does not induce significant hematological orhepatic toxic side effects in an in vivo murine model. In someembodiments, hematological side effects are assessed by measuring meanneutrophil, mean white blood cell or mean platelet counts. In someembodiments, hepatic toxic side effects are assessed by measuring serumaspartate transaminase (AST), serum alanine transaminase (ALT), and/orserum albumin levels. In some embodiments, an alpha polyglutamatedAntifolate composition provided herein does not significantly decreasemean neutrophil, mean white blood cell or mean platelet counts. In someembodiments, an alpha polyglutamated Antifolate composition providedherein does not significantly increase serum aspartate transaminase(AST) and serum alanine transaminase (ALT) levels. In some embodiments,an alpha polyglutamated Antifolate composition provided herein does notsignificantly decrease serum albumin levels. In some embodiments, the invivo assay comprises administering 40 mg/kg or 80 mg/kg of the alphapolyglutamated Antifolate composition once weekly for 4 weeks. In someembodiments, the alpha polyglutamated Antifolate is a hexaglutamatedAntifolate.

In some embodiments, the alpha polyglutamated Antifolate compositions donot contain a fluorine atom. In some embodiments, the alphapolyglutamated Antifolate compositions do not contain a 4-fluoroglutamylgroup.

Alpha polyglutamated Antifolate (αPANTIFOL) compositions and their usesare further described in each of Intl. Appl. Nos. PCT/US2017/046666 andPCT/US2017/046667, and U.S. Patent Appl. Nos. 62/630,820, 62/627,716,62/627,731, 62/630,671, 62/630,825, 62/630,629, 62/630,634, 62/630,728,62/630,637, 62/630,744, 62/583,432, 62/627,714, 62/627,741, and62/627,703, the disclosure of each of which is herein incorporated byreference in its entirety.

A. Alpha Polyglutamated Antifolate Analogs and Derivatives

The disclosure also encompasses alpha polyglutamated Antifolatederivatives and analogs. The compositions and methods disclosed hereinare envisioned to apply to any and every known derivative or analog ofan Antifolate that is polyglutamated. In some embodiments, the analogcorresponds to a modified form of the Antifolate wherein the glutamylgroup of the Antifolate is not linked to the remainder of the Antifolatemolecule through a gamma peptide linkage. In some embodiments, theanalog is a variant form of the Antifolate wherein the glutamyl group inthe Antifolate is in the D-form. In some embodiments, the polyglutamatedform of the Antifolate, or polyglutamated Antifolate analog orderivative, is not fluorinated.

In some embodiments, the Antifolate is selected from: an indoline ringand modified ornithine-bearing methotrexate derivative, an indoline ringand modified glutamic acid-bearing methotrexate derivative, analkyl-substituted benzene ring C bearing methotrexate derivative, abenzoxazine moiety-bearing methotrexate derivative, a benzothiazinemoiety-bearing methotrexate derivative, a 10-deazaminopterin analog, a5-deazaminopterin methotrexate analog, a 5,10-dideazaminopterinmethotrexate analog, a indoline moiety-bearing methotrexate derivative,a lipophilic amide methotrexate derivative, aL-threo-(2S,4S)-4-fluoro-glutamic acid containing methotrexate analog, aDL-3,3-difluoroglutamic acid-containing methotrexate analog, amethotrexate tetrahydroquinazoline analog, a N-(ac-aminoacyl)methotrexate derivative, a biotin methotrexate derivative, a D-glutamicacid methotrexate analog, a D-erythrou, threo-4-fluoroglutamic acidmethotrexate analog, β,γ-methano methotrexate analog, a10-deazaminopterin (10-EDAM) analog, a γ-tetrazole methotrexate analog,a N-(L-α-aminoacyl) methotrexate derivative, a meta isomer ofaminopterin, an ortho isomer of aminopterin, ahydroxymethylmethotrexate, a γ-fluoromethotrexate, a polyglutamylmethotrexate derivative, a gem-diphosphonate methotrexate analog (see,e.g., WO1988/06158, the contents of which is herein incorporated byreference in its entirety), a α-substituted methotrexate analog, aγ-substituted methotrexate analog, a 5-methyl-5-deaza methotrexateanalog (see. e.g., U.S. Pat. No. 4,725,687, the contents of each ofwhich is herein incorporated by reference in its entirety), an Ndelta-acyl-N α-(4-amino-4-deoxypteroyl)-L-ornithine derivative, a8-deaza methotrexate analog, an acivicin methotrexate analog, apolymeric platinol methotrexate derivative, amethotrexate-γ-dimyristoylphophatidylethanolamine, a methotrexatepolyglutamate analog, a poly-γ-glutamyl methotrexate derivative, adeoxyuridylate methotrexate derivative, a iodoacetyl lysine methotrexateanalog, a 2,omega.-diaminoalkanoid acid-containing methotrexate analog,a polyglutamate methotrexate derivative, a 5-methyl-5-deaza analog, aquinazoline methotrexate analog, a pyrazine methotrexate analog, acysteic or homocysteic acid methotrexate analog (see, e.g., U.S. Pat.No. 4,490,529, and EPA 0142220, the contents of each of which is hereinincorporated by reference in its entirety), a γ-tert-butyl methotrexateester, a fluorinated methotrexate analog, a folate methotrexate analog,a phosphonoglutamic acid analog, a poly (L-lysine) methotrexateconjugate, a dilysine or trilysine methotrexate derivate, a7-hydroxymethotrexate, a poly-γ-glutamyl methotrexate analog, a3′,5′-dichloromethotrexate, a diazoketone or chloromethylketonemethotrexate analog, a 10-propargylaminopterin, an alkyl methotrexatehomologs, a lectin derivative of methotrexate, a polyglutamatemethotrexate derivative, a halogenated methotrexate derivative, a8-alkyl-7,8-dihydro analog, a 7-methyl methotrexate derivative, adichloromethotrexate, a lipophilic methotrexate derivative, a3′,5′-dichloromethotrexate, a deaza amethopterin analog, and MX068, or astereoisomer thereof.

In additional embodiments, the alpha polyglutamated Antifolatederivative or analog has a variant polyglutamate chain. In someembodiments, the polyglutamate chain contains one or more natural orsynthetic residues other than glutamate. In some embodiments, thepolyglutamate chain contains one or more glutamyl groups that do notcontain an amide linkage. In other embodiments, one or more of theglutamyl groups of the polyglutamate chain is derivatized.

B. αANTIFOL-PG Synthesis

The Antifolate polyglutamate compositions provided herein may beobtained by following synthetic procedures known in the art. Proceduresfor synthesizing Antifolate (including different pharmaceuticallyacceptable salts or acids (e.g., Antifolate disodium) and crystallineand amorphous forms) and intermediates for synthesizing Antifolateinclude but are not limited to those described in U.S. Pat. Nos.2,512,572; 3,892,801; 3,989,703; 4,057,548; 4,067,867; 4,079,056;4,080,325; 4,106,488; 4,136,101; 4,224,446; 4,306,064; 4,374,987;4,421,913; 4,558,690; 4,662,359; and 4,767,859; and Calvert, Semin.Oncol. 26:3-10 (1999)).

The Antifolate polyglutamate compositions provided herein may beobtained by following synthetic procedures using available reagents andsynthetic intermediates. The addition of glutamyl residue(s) to theglutamyl residue of the Antifolate can be accomplished using syntheticprocedures known in the art. In some embodiments, glutamyl residues areadded serially to the glutamyl residue of the Antifolate. In additionalembodiments, polyglutamates are added to the glutamyl reside of theAntifolate using “click chemistry” methods or other bioconjugatechemistries known to those in the art. Alternatively, a peptide ofglutamyl residues can be generated of the desired length and added to aprecursor of the Antifolate which does not have a glutamyl residue. Thepeptide can be produced using synthetic procedures known in the art. Insome embodiments, an initial glutamyl residue is bonded to wang resinand additional glutamyl residues are added serially via solid phasepeptide synthesis using F-moc chemistry. After the final glutamylresidue is added the Antifolate precursor is coupled to the peptide andthe molecule is cleaved from the resin.

C. Alpha Polyglutamated Antifolate Complexes

The inventors have surprisingly found that polyglutamated Antifolatessuch as polyglutamated pemetrexed are able to form complexes with othercompositions including therapeutic agents, including cytotoxic compoundssuch as platinum-based compounds. Accordingly, in some embodiments, thedisclosure provides a complex of a αPANTIFOL (e.g., a αPANTIFOLdisclosed herein) and a therapeutic agent or a salt or acid thereof. Insome embodiments, the polyglutamated Antifolate is a αPANTIFOL describedin Section II, or a salt or acid thereof. In some embodiments, thedisclosure provides a complex of a αPANTIFOL according to any of[1]-[12] of the Detailed Description and a therapeutic agent or a saltor acid thereof. In some embodiments, the αPANTIFOL/complex compriseαPANTIFOL and a therapeutic agent. In some embodiments, the therapeuticagent is a cytotoxic compound such as a chemotherapeutic agent. Infurther embodiments, the αPANTIFOL/complex contains a platinum-baseddrug such as platinum-based chemotherapeutic agent (e.g., cisplatin,carboplatin and oxaliplatin). In other embodiments, theαPANTIFOL/complex contains a taxane-based chemotherapeutic agent (e.g.,paclitaxel and docetaxel). In other embodiments, the αPANTIFOL/complexcontains a cyclodextrin. In further embodiments, the αPANTIFOL/complexis encapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In further embodiments, the αPANTIFOL/therapeutic agent complexcomprises one or more αPANTIFOL containing 2-150, 2-100, 2-75, 2-50,2-24, 2-30, 2-20, 2-19, 2-15, 2-10, or 2-5, glutamyl groups. In someembodiments, the αPANTIFOL/therapeutic agent complex comprises one ormore αPANTIFOL containing 3-10, 3-9, 3-8, or 3-7, glutamyl groups, orany range therein between. In other embodiments, theαPANTIFOL/therapeutic agent complex comprises one or more αPANTIFOLcontaining 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5, glutamyl groups, or anyrange therein between. In one particular embodiment, the complexcomprises one or more αPANTIFOL containing 3-10 glutamyl groups. Infurther embodiments, the αPANTIFOL/therapeutic agent complex comprisesone or more αPANTIFOL containing 3-7 glutamyl groups. In anotherembodiment, the αPANTIFOL/therapeutic agent complex comprises one ormore αPANTIFOL containing 5 glutamyl groups. In another embodiment, theαPANTIFOL/therapeutic agent complex comprises one or more αPANTIFOLcontaining 6 glutamyl groups. In some embodiments, the therapeutic agentis a cytotoxic compound or a salt or acid thereof. In a furtherembodiment, the therapeutic agent is a chemotherapeutic agent or a saltor acid thereof. In another embodiment, the therapeutic agent is aplatinum-based drug. In another embodiment, the therapeutic agent is ataxane-based drug. In additional embodiments, the molar ratio ofαPANTIFOL/therapeutic agent in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of αPANTIFOL/therapeutic agent in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 10:1. In someembodiments, the αPANTIFOL/therapeutic agent complex is encapsulated ina liposome (e.g., as described herein or otherwise known in the art). Insome embodiments, the molar ratio of αPANTIFOL/therapeutic agent in thecomplex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50. In some embodiments, the molar ratio of αPANTIFOL/therapeuticagent in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In some embodiments, the αPANTIFOL/therapeuticagent complex is encapsulated in a liposome (e.g., as described hereinor otherwise known in the art). In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In an alternative embodiment, the αPANTIFOL complex comprises αPANTIFOLand cyclodextrin. In some embodiments, the αPANTIFOL complex comprises aαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the αPANTIFOL complex comprises an Antifolatedescribed in Section II. In some embodiments, the molar ratio ofαPANTIFOL (e.g., αPANTIFOL salt)/cyclodextrin in the complex is in therange 1-20:1, or any range therein between. In some embodiments, themolar ratio of αPANTIFOL/cyclodextrin in the complex is in the range1-10:1, or any range therein between. In further embodiments, the molarratio of αPANTIFOL/cyclodextrin in the complex is in the range 2-8:1, orany range therein between. In some embodiments, the molar ratio ofαPANTIFOL/cyclodextrin in the complex is: 1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, or 20:1. In some embodiments, the molar ratio ofαPANTIFOL/cyclodextrin in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof αPANTIFOL/cyclodextrin in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of αPANTIFOL/cyclodextrin in the complex is: 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/cyclodextrin in the complex is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, theαPANTIFOL/cyclodextrin complex is encapsulated in a liposome. In someembodiments, the liposome is an Lp-αPANTIFOL according to any of[13]-[72] of the Detailed Description.

In some embodiments, the disclosure provides a composition comprising aαPANTIFOL/platinum-based chemotherapeutic agent complex. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In some embodiments, the platinum-based chemotherapeutic agent isselected from: cisplatin, carboplatin, and oxaliplatin, or a salt oracid thereof. In other embodiments, the αPANTIFOL/platinum-basedchemotherapeutic agent complex comprises an analog of a cisplatin,carboplatin, oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is in the range 1-20:1, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is 11:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In someembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio ofαPANTIFOL/platinum-based chemotherapeutic agent in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/platinum-based agent in thecomplex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments,the molar ratio of αPANTIFOL/platinum-based agent in the complex is:1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPANTIFOL//platinum-based agent complex is encapsulatedin a liposome. In some embodiments, the liposome is an Lp-αPANTIFOLaccording to any of [13]-[72] of the Detailed Description.

In additional embodiments, the αPANTIFOL/platinum-based chemotherapeuticagent complex comprises an analog of a cisplatin, carboplatin,oxaliplatin, or a salt or acid thereof. In some embodiments, the complexcomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the αPANTIFOL complex comprises apolyglutamated Antifolate described in Section II. In some embodiments,the molar ratio of αPANTIFOL/platinum-based analog in the complex is inthe range 1-20:1, or any range therein between. In some embodiments, themolar ratio of αPANTIFOL/platinum-based analog in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of αPANTIFOL/platinum-based agent in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPANTIFOL/platinum-based analog in the complex is 11:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the molarratio of αPANTIFOL/platinum-based analog in the complex is 11:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments,the molar ratio of αPANTIFOL/platinum-based agent in the complex is:1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the molarratio of αPANTIFOL/platinum-based agent in the complex is: 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the αPANTIFOL//platinum-based analog complex is encapsulated in aliposome. In some embodiments, the liposome is an Lp-αPANTIFOL accordingto any of [13]-[72] of the Detailed Description.

In further embodiments, the disclosure provides a complex containingαPANTIFOL and cisplatin or a salt or acid thereof. In some embodiments,the complex comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the αPANTIFOL complexcomprises an Antifolate described in Section II. In some embodiments,the molar ratio of αPANTIFOL/cisplatin (or cisplatin salt or acid) inthe complex is in the range 1-20:1, or any range therein between. Insome embodiments, the molar ratio of αPANTIFOL/cisplatin (or cisplatinsalt or acid) in the complex is in the range 1-10:1, or any rangetherein between. In further embodiments, the molar ratio ofαPANTIFOL/cisplatin (or cisplatin salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPANTIFOL/cisplatin (or cisplatin salt or acid) in thecomplex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments,the molar ratio of αPANTIFOL/cisplatin (or cisplatin salt or acid) inthe complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In some embodiments, the molar ratio of αPANTIFOL/cisplatin(or cisplatin salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/cisplatin (or cisplatin salt or acid) in the complex is: 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPANTIFOL//cisplatin (or cisplatin salt or acid)complex is encapsulated in a liposome. In some embodiments, the liposomeis an Lp-αPANTIFOL according to any of [13]-[72] of the DetailedDescription.

In another embodiment, the disclosure provides a complex containingαPANTIFOL and carboplatin or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II,herein. In some embodiments, the molar ratio of αPANTIFOL/carboplatin(or carboplatin salt or acid) in the complex is in the range 1-20:1, orany range therein between. In further embodiments, the molar ratio ofαPANTIFOL/carboplatin (or carboplatin salt or acid) in the complex is inthe range 1-10:1, or any range therein between. In further embodiments,the molar ratio of αPANTIFOL/carboplatin (or carboplatin salt or acid)in the complex is in the range 2-8:1, or any range therein between. Insome embodiments, the molar ratio of αPANTIFOL/carboplatin (orcarboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, or 20:1. In some embodiments, the molar ratio ofαPANTIFOL/carboplatin (or carboplatin salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In someembodiments, the molar ratio of αPANTIFOL/carboplatin in the complex is:1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the molarratio of αPANTIFOL/carboplatin in the complex is: 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, theαPANTIFOL/carboplatin (or carboplatin salt or acid) complex isencapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In another embodiment, the disclosure provides a complex containingαPANTIFOL and oxaliplatin, or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In some embodiments, the molar ratio of αPANTIFOL/oxaliplatin (oroxaliplatin salt or acid) in the complex is in the range 1-20:1, or anyrange therein between. In further embodiments, the molar ratio ofαPANTIFOL/oxaliplatin (or oxaliplatin salt or acid) in the complex is inthe range 1-10:1, or any range therein between. In further embodiments,the molar ratio of αPANTIFOL/oxaliplatin (or oxaliplatin salt or acid)in the complex is in the range 2-8:1, or any range therein between. Insome embodiments, the molar ratio of αPANTIFOL/oxaliplatin (oroxaliplatin salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/oxaliplatin (or oxaliplatin salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In someembodiments, the molar ratio of αPANTIFOL/oxaliplatin (or oxaliplatinsalt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. Insome embodiments, the molar ratio of αPANTIFOL/oxaliplatin (oroxaliplatin salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, theαPANTIFOL/oxaliplatin (or oxaliplatin salt or acid) complex isencapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and a platinum-based chemotherapeutic agent (“platinum”)selected from: nedaplatin, heptaplatin, lobaplatin, stratoplatin,paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin,picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and dedaplatin, or a salt or acid thereof. In otherembodiments, the αPANTIFOL/platinum-based chemotherapeutic agent complexcomprises an analog of nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, or dedaplatin, or a salt or acid thereof. Insome embodiments, the molar ratio of αPANTIFOL/platinum (or platinumsalt or acid) in the complex is in the range 1-20:1, or any rangetherein between. In some embodiments, the complex comprises a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the αPANTIFOL complex comprises a polyglutamated Antifolatedescribed in Section II. In further embodiments, the molar ratio ofαPANTIFOL/platinum (or platinum salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of αPANTIFOL/platinum (or platinum salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/platinum (or platinum salt oracid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Insome embodiments, the molar ratio of αPANTIFOL/platinum (or platinumsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In some embodiments, the molar ratio ofαPANTIFOL/platinum (or platinum salt or acid) in the complex is: 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/platinum (or platinum salt or acid) in the complex is: 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPANTIFOL/platinum (or salt or acid or analog thereof)complex is encapsulated in a liposome. In some embodiments, the liposomeis an Lp-αPANTIFOL according to any of [13]-[72] of the DetailedDescription.

In some embodiments, the disclosure provides a composition comprising aαPANTIFOL/taxane-based chemotherapeutic agent (taxane) complex. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In some embodiments, the taxane-based chemotherapeutic agent is selectedfrom: paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), andcabazitaxel (CTX), or a salt or acid thereof. In some embodiments, themolar ratio of αPANTIFOL/taxane-based agent in the complex is in therange 1-20:1, or any range therein between. In further embodiments, themolar ratio of αPANTIFOL/taxane (or taxane salt or acid) in the complexis in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of αPANTIFOL/taxane (or taxane salt oracid) in the complex is in the range 2-8:1, or any range thereinbetween. In some embodiments, the molar ratio of αPANTIFOL/taxane (ortaxane salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, or 20:1. In some embodiments, the molar ratio of αPANTIFOL/taxane(or taxane salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the molar ratio ofαPANTIFOL/taxane (or taxane salt or acid) in the complex is: 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/taxane (or taxane salt or acid) in the complex is: 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the αPANTIFOL/taxane (or taxane salt or acid) agent complex isencapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and paclitaxel (PTX), or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In other embodiments, the αPANTIFOL/paclitaxel (or paclitaxel salt oracid) complex comprises an analog of paclitaxel (PTX), or a salt or acidthereof. In some embodiments, the molar ratio of αPANTIFOL/paclitaxel(or paclitaxel salt or acid) in the complex is in the range 1-20:1, orany range therein between. In further embodiments, the molar ratio ofαPANTIFOL/paclitaxel (or paclitaxel salt or acid) in the complex is inthe range 1-10:1, or any range therein between. In further embodiments,the molar ratio of αPANTIFOL/paclitaxel (or paclitaxel salt or acid) inthe complex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/paclitaxel (or paclitaxel saltor acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Insome embodiments, the molar ratio of αPANTIFOL/paclitaxel (or paclitaxelsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In some embodiments, the molar ratio ofαPANTIFOL/paclitaxel (or paclitaxel salt or acid) in the complex is:1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the molarratio of αPANTIFOL/paclitaxel (or paclitaxel salt or acid) in thecomplex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the αPANTIFOL/paclitaxel (or paclitaxel salt oracid) complex is encapsulated in a liposome. In some embodiments, theliposome is an Lp-αPANTIFOL according to any of [13]-[72] of theDetailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and docetaxel (DTX), or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description.

In some embodiments, the αPANTIFOL complex comprises a polyglutamatedAntifolate described in Section II. In other embodiments, theαPANTIFOL/docetaxel complex comprises an analog of docetaxel (DTX), or asalt or acid thereof. In some embodiments, the molar ratio ofαPANTIFOL/docetaxel (or docetaxel salt or acid) in the complex is in therange 1-20:1, or any range therein between. In some embodiments, themolar ratio of αPANTIFOL/docetaxel (or docetaxel salt or acid) in thecomplex is in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of αPANTIFOL/docetaxel (or docetaxel saltor acid) in the complex is in the range 2-8:1, or any range thereinbetween. In some embodiments, the molar ratio of αPANTIFOL/docetaxel (ordocetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, or 20:1. In some embodiments, the molar ratio ofαPANTIFOL/docetaxel (or docetaxel salt or acid) in the complex is 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In someembodiments, the molar ratio of αPANTIFOL/docetaxel (or docetaxel saltor acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. Insome embodiments, the molar ratio of αPANTIFOL/docetaxel (or docetaxelsalt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, the αPANTIFOL/docetaxel(or docetaxel salt or acid) complex is encapsulated in a liposome. Insome embodiments, the liposome is an Lp-αPANTIFOL according to any of[13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and larotaxel (LTX), or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In some embodiments, the molar ratio of αPANTIFOL/larotaxel (orlarotaxel salt or acid) in the complex is in the range 1-20:1, or anyrange therein between. In further embodiments, the molar ratio ofαPANTIFOL/larotaxel (or larotaxel salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of αPANTIFOL/larotaxel (or larotaxel salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPANTIFOL/larotaxel (or larotaxel saltor acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Insome embodiments, the molar ratio of αPANTIFOL/larotaxel (or larotaxelsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In some embodiments, the molar ratio ofαPANTIFOL/larotaxel (or larotaxel salt or acid) in the complex is: 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the molar ratio ofαPANTIFOL/larotaxel (or larotaxel salt or acid) in the complex is: 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPANTIFOL/larotaxel (or larotaxel salt or acid) complexis encapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and cabazitaxel (CTX), or a salt or acid thereof. In someembodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.In some embodiments, the molar ratio of αPANTIFOL/cabazitaxel (orcabazitaxel salt or acid) in the complex is in the range 1-20:1, or anyrange therein between. In further embodiments, the molar ratio ofαPANTIFOL/cabazitaxel (or cabazitaxel salt or acid) in the complex is inthe range 1-10:1, or any range therein between. In further embodiments,the molar ratio of αPANTIFOL/cabazitaxel (or cabazitaxel salt or acid)in the complex is in the range 2-8:1, or any range therein between. Insome embodiments, the molar ratio of αPANTIFOL/cabazitaxel (orcabazitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, or 20:1. In some embodiments, the molar ratio ofαPANTIFOL/cabazitaxel (or cabazitaxel salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In someembodiments, the molar ratio of αPANTIFOL/cabazitaxel (or cabazitaxelsalt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. Insome embodiments, the molar ratio of αPANTIFOL/cabazitaxel (orcabazitaxel salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, theαPANTIFOL/cabazitaxel (or cabazitaxel salt or acid) complex isencapsulated in a liposome. In some embodiments, the liposome is anLp-αPANTIFOL according to any of [13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex comprisingαPANTIFOL and another anti-metabolite, or a salt or acid thereof. Insome embodiments, the complex comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the αPANTIFOLcomplex comprises a polyglutamated Antifolate described in Section II.An anti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. In some embodiments, the disclosure provides a complexcomprising αPANTIFOL and Antifolate (ANTIFOL), or a salt or acidthereof. In some embodiments, the disclosure provides a complexcomprising αPANTIFOL and an anti-metabolite selected from, gemcitabine,fluorouracil, capecitabine, an antifolate (e.g., Antifolate,raltitrexed), tegafur, cytosine arabinoside, thioguanine, 5-azacytidine,6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabinephosphate, and cladribine, as well as pharmaceutically acceptable saltor acids, acids, or derivatives of any of these. In some embodiments,the molar ratio of αPANTIFOL/anti-metabolite (or anti-metabolite salt oracid, or prodrug) in the complex is in the range 1-20:1, or any rangetherein between. In further embodiments, the molar ratio ofαPANTIFOL/anti-metabolite (or anti-metabolite salt or acid, or prodrug)in the complex is in the range 1-10:1, or any range therein between. Infurther embodiments, the molar ratio of αPANTIFOL/anti-metabolite (oranti-metabolite salt or acid, or prodrug) in the complex is in the range2-8:1, or any range therein between. In some embodiments, the molarratio of αPANTIFOL/anti-metabolite (or anti-metabolite salt or acid, orprodrug) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Insome embodiments, the molar ratio of αPANTIFOL/anti-metabolite (oranti-metabolite salt or acid, or prodrug) in the complex is 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments,the molar ratio of αPANTIFOL/anti-metabolite (or anti-metabolite salt oracid, or prodrug) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or1:20. In some embodiments, the molar ratio of αPANTIFOL/anti-metabolite(or anti-metabolite salt or acid, or prodrug) in the complex is 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPANTIFOL/anti-metabolite (or anti-metabolite salt oracid, or prodrug) complex is encapsulated in a liposome. In someembodiments, the liposome is an Lp-αPANTIFOL according to any of[13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a complex ofαPANTIFOL (e.g., a αPANTIFOL disclosed herein) and a cyclodextrin.Cyclodextrins (CDs) are groups of cyclic oligosaccharides which havebeen shown to improve physicochemical properties of many drugs throughformation of complexes. CDs are cyclic oligosaccharides composed ofseveral D-glucose units linked by α-(1, 4) bonds. This cyclicconfiguration provides a hydrophobic internal cavity and gives the CDs atruncated cone shape. Many hydroxyl groups are situated on the edges ofthe ring which make the CDs both lipophilic and soluble in water. As aresult, CDs are able to form complexes with a wide variety ofhydrophobic agents, and thus change the physical-chemical properties ofthese complexed agents. In some embodiments, the complex comprises aαPANTIFOL according to any of [1]-[12] of the Detailed Description.

The terms “cyclodextrin” or “CD” unless otherwise specified herein,refer generally to a parent or derivatized cyclic oligosaccharidecontaining a variable number of (α-1,4)-linked D-glucopyranoside unitsthat is able to form a complex with a Antifolate-PG. Each cyclodextringlucopyranoside subunit has secondary hydroxyl groups at the 2 and 3positions and a primary hydroxyl group at the 6-position. The terms“parent”, “underivatized”, or “inert”, cyclodextrin refer to acyclodextrin containing D-glucopyranoside units having the basic formulaC₆H₁₂O₆ and a glucose structure without any additional chemicalsubstitutions (e.g., α-cyclodextrin consisting of 6 D-glucopyranosideunits, a β-cyclodextrin consisting of 7 D-glucopyranoside units, and aγ-cyclodextrin cyclodextrin consisting of 8 D-glucopyranoside units).The physical and chemical properties of a parent cyclodextrin can bemodified by derivatizing the hydroxyl groups with other functionalgroups. Any substance located within the cyclodextrin internal phase issaid to be “complexed” with the cyclodextrin, or to have formed acomplex (inclusion complex) with the cyclodextrin.

As used herein, there are no particular limitations on the cyclodextrincomponent of the αPANTIFOL/cyclodextrin complexes so long as thecyclodextrins can form complexes with the αPANTIFOL. In particularembodiments, the cyclodextrins have been derivatized to bear ionizable(e.g., weakly basic and/or weakly acidic) functional groups tofacilitate complex formation with αPANTIFOL and/or liposomeencapsulation.

Modifications of the hydroxyl groups of cyclodextrins, such as thosefacing away from the cyclodextrin interior phase, with ionizablechemical groups is known to facilitate the loading of cyclodextrins andtherapeutic agents complexed with the cyclodextrins. In someembodiments, the cyclodextrin of the αPANTIFOL/cyclodextrin complex hasat least 2, 3, 4, 5, 6, 6, 7, 8, 9, or 10, hydroxyl group substitutedwith an ionizable chemical group. The term “charged cyclodextrin” refersto a cyclodextrin having one or more of its hydroxyl groups substitutedwith a charged moiety. Such a moiety can itself be a charged group or itcan comprise an organic moiety (e.g., a C₁-C₆ alkyl or C₁-C₆ alkyl ethermoiety) substituted with one or more charged moieties.

In some embodiments, the “ionizable” or “charged” moieties of a CDderivative are weakly ionizable. Weakly ionizable moieties are thosethat are either weakly basic or weakly acidic. Weakly basic functionalgroups (W) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0,7.5-8.0, and any range in between inclusive according to CH3-W.Similarly, weakly acidic functional groups (X) have a log dissociationconstant (pKa) of between about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0,5.0-5.5, and any range in between inclusive according to CH3-X.Representative anionic moieties include, without limitation,carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkylether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate,phosphonate, sulfonate, nitrate, and borate groups. Representativecationic moieties include, without limitation, amino, guanidine, andquarternary ammonium groups.

In another embodiment, the derivatized cyclodextrin is a “polyanion” or“polycation.” A polyanion is a derivatized cyclodextrin having more thanone negatively charged group resulting in net a negative ionic charge ofmore than two units. A polycation is a derivatized cyclodextrin havingmore than one positively charged group resulting in net positive ioniccharger of more than two units.

In another embodiment, the derivatized cyclodextrin is a “chargeableamphiphile.” By “chargeable” is meant that the amphiphile has a pK inthe range pH 4 to pH 8 or 8.5. A chargeable amphiphile may therefore bea weak acid or base. By “amphoteric” herein is meant a derivatizedcyclodextrin having a ionizable groups of both anionic and cationiccharacter wherein: (a) at least one, and optionally both, of the cationand anionic amphiphiles is chargeable, having at least one charged groupwith a pK between 4 and 8 to 8.5, (b) the cationic charge prevails at pH4, and (c) the anionic charge prevails at pH 8 to 8.5.

In some embodiments, the “ionizable” or “charged” derivatizedcyclodextrin as a whole, whether polyionic, amphiphilic, or otherwise,are weakly ionizable (i.e., have a pKai of between about 4.0-8.5,4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in between inclusive).

Any one, some, or all hydroxyl groups of any one, some or allα-D-glucopyranoside units of a cyclodextrin can be modified to anionizable chemical group as described herein. Since each cyclodextrinhydroxyl group differs in chemical reactivity, reaction with a modifyingmoiety can produce an amorphous mixture of positional and opticalisomers. Alternatively, certain chemistry can allow for pre-modifiedα-D-glucopyranoside units to be reacted to form uniform products.

The aggregate substitution that occurs for cyclodextrin derivatives in amixture is described by a term referred to as the degree ofsubstitution. For example, a 6-ethylenediamino-β-cyclodextrin with adegree of substitution of seven would be composed of a distribution ofisomers of 6-ethylenediamino-β-cyclodextrin in which the average numberof ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin moleculeis seven. The degree of substitution for a cyclodextrin derivativemixture can routinely be determined using mass spectrometry or nuclearmagnetic resonance spectroscopy.

In one embodiment, at least one hydroxyl moieties facing away from thecyclodextrin interior is substituted with an ionizable chemical group.For example, the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and allthree of C2-C3-C6 hydroxyls of at least one α-D-glucopyranoside unit aresubstituted with an ionizable chemical group. Any such combination ofhydroxyls can similarly be combined with at least two, three, four,five, six, seven, eight, nine, ten, eleven, up to all of thealpha-D-glucopyranoside units in the modified cyclodextrin as well as incombination with any degree of substitution described herein. One suchderivative is a sulfoalkyl ether cyclodextrin (SAE-CD). Sulfobutyl etherderivatives of beta cyclodextrin (SBE-β-CD) have been demonstrated tohave significantly improved aqueous solubility compared to the parentcyclodextrin.

Additional cyclodextrin derivatives that may be complexed withtherapeutic agents in the disclosed liposome compositions includesugammadex or Org-25969, in which the 6-hydroxy groups on γ-CD have beenreplaced by carboxythio acetate ether linkages, and hydroxybutenyl-β-CD.Alternative forms of cyclodextrin include: 2,6-Di-O-methyl-β-CD (DIMEB),2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomlymethylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin(SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylatedbeta-cyclodextrin sodium salt, (2-Hydroxypropyl)-alpha-cyclodextrin,(2-Hydroxypropyl)-beta-cyclodextrin, (2-Hydroxypropyl)-γ-cyclodextrin,2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 Heptakis),2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB Heptakis),methyl-beta-cyclodextrin, octakis (6-deoxy-6-iodo)-γ-cyclodexrin, and,octakis (6-deoxy-6-bromo)-gamma-cyclodexrin.

In some embodiments, the cyclodextrin(s) has a high solubility in waterin order to facilitate entrapment of a larger amount of the cyclodextrinin the liposome internal phase. In some embodiments, the watersolubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL,40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL orhigher. In some embodiments, the water solubility of the cyclodextrin(s)is within a range of 10-150 mg/mL, 20-100 mg/mL 20-75 mg/mL, and anyrange in between inclusive.

In some embodiments, a large association constant between thecyclodextrin and the αPANTIFOL and/or other therapeutic agent complexedwith cyclodextrin is preferable and can be obtained by selecting thenumber of glucose units in the cyclodextrin based on the size of thetherapeutic agent (see, for example, Albers et al., Crit. Rev. Therap.Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol.36:30-42 (2008). When the association constant depends on pH, thecyclodextrin can be selected such that the association constant becomeslarge at the pH of the liposome internal phase. As a result, thesolubility (nominal solubility) of the therapeutic agent in the presenceof cyclodextrin can be further improved. In some embodiments, theassociation constant of the cyclodextrin with the therapeutic agent is100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher. In someembodiments, the association constant of the cyclodextrin with thetherapeutic agent is in the range 100-1, 200, 200-1,000, 300-750, andany range therein between.

In some embodiments, the cyclodextrin of the αPANTIFOL/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is underivatized.

In some embodiments, the cyclodextrin of the αPANTIFOL/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is derivatized. Infurther embodiments, the cyclodextrin derivative of the complex has thestructure of Formula I:

wherein: n is 4, 5, or 6;wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—H, a straight chain or branched C₁-C₈-alkylene group, or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉, is a straight-chain orbranched C₁-C₈-alkylene (e.g., C₁-C₈-(alkylene)-SO₃ ⁻ group);

In some embodiments, the cyclodextrin derivative of theαPANTIFOL/cyclodextrin complex and/or cyclodextrin/therapeutic agentcomplex has the structure of Formula II:

wherein: n is 4, 5, or 6;wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻ group; wherein at least one of R₁ andR₂ is independently a —O—(C₂-C₆ alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃,S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, a pharmaceuticallyacceptable cation. In further embodiments, the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺² and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine. In someembodiments, at least one of R1 and R2 is independently a —O—(C2-C6alkylene)-SO3- group that is a —O—(CH₂)_(m)SO3- group, wherein m is 2 to6, preferably 2 to 4, (e.g., —O—CH2CH2CH2S03- or —O—CH2CH2CH2CH2S03-);and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, H ora pharmaceutically cation which includes for example, alkali metals(e.g., Li⁺, Na⁺, K⁺) alkaline earth metals (e.g., Ca⁺², Mg⁺²), ammoniumions and amine cations such as the cations of (C1-C6)-alkylamines,piperidine, pyrazine, (C₁-C₆)-alkanolamine and(C₄-C₈)-cycloalkanolamine:

In some embodiments, a cyclodextrin derivative of theαPANTIFOL/cyclodextrin complex and/or cyclodextrin/therapeutic agentcomplex is a cyclodextrin disclosed in U.S. Pat. Nos. 6,133,248,5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; andIntl. Appl. Publ. No. WO 02005/117911, the contents each of which isherein incorporated by reference in its priority.

In some embodiments, the cyclodextrin derivative of theαPANTIFOL/cyclodextrin complex and/or cyclodextrin/therapeutic agentcomplex is a sulfoalkyl ether cyclodextrin. In some embodiments, thecyclodextrin derivative of complex is a sulfobutyl ether-3-cyclodextrinsuch as CAPTISOL® (CyDex Pharma. Inc., Lenexa, Kans.). Methods forpreparing sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ethercyclodextrins are known in the art.

In some embodiments, the cyclodextrin derivative in of theαPANTIFOL/cyclodextrin complex and/or cyclodextrin/therapeutic agentcomplex is a compound of Formula III:

wherein R equals:

-   -   (a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0;    -   (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0;    -   (c) (H)_(21-X) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0; or    -   (d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0.

In additional embodiments, the αPANTIFOL/cyclodextrin complex and/orcyclodextrin/therapeutic agent complex is encapsulated in a liposome(e.g., as described herein or otherwise known in the art).

III. αPANTIFOL Delivery Vehicles

In alternative embodiments, the disclosure provides αPANTIFOL deliverysystems and their use to deliver a payload of αPANTIFOL to a cell orcells in vitro or in vivo. In some embodiments, αPANTIFOL is complexedwith or incorporated into a delivery vehicle. Such delivery vehicles areknown in the art and include, but are not limited to, liposomes,lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs suchas Antibody-αPANTIFOL conjugates), cellular components, cyclicoligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipidnanoparticles, biodegradable nanoparticles, and core-shellnanoparticles), lipoprotein particles, and combinations thereof. Inparticular embodiments, the delivery vehicle is a liposome. In otherparticular embodiments, the delivery vehicle is an antibody or anantigen binding antibody fragment. In some embodiments, the αPANTIFOLdelivery system comprises a αPANTIFOL according to any of [1]-[12] ofthe Detailed Description.

A. Liposomes

In some embodiments, the disclosure provides liposomal compositions thatcomprise a liposome encapsulating (i.e., filled with) an alphapolyglutamated Antifolate (e.g., a αPANTIFOL disclosed herein). In someembodiments, the liposomal composition comprises a αPANTIFOL accordingto any of [1]-[12] of the Detailed Description. In some embodiments, theliposomal composition comprises a polyglutamated Antifolate described inSection II. In some embodiments, a liposome in the liposomal compositioncomprises a αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups (including the glutamyl group of the Antifolate). Insome embodiments, the liposomal composition contains a liposomeaccording to any of [13]-[72] of the Detailed Description. In someembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises two or more glutamyl groups in the L-form. In otherembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises a glutamyl group in the D-form. In further embodiments, thealpha polyglutamated Antifolate in the Lp-αPANTIFOL comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form. Inadditional embodiments, the alpha polyglutamated Antifolate in theLp-αPANTIFOL comprises two or more glutamyl groups that have a gammacarboxyl linkage. In some embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL comprises at least one glutamyl groupthat has both an alpha carboxyl linkage and a gamma carboxyl linkage. Insome embodiments, the liposomal composition comprises a liposomecomprising and α pentaglutamated Antifolate. In further embodiments, theliposome comprises an L-α pentaglutamated Antifolate, a D-αpentaglutamated Antifolate, or an L- and D-α pentaglutamated Antifolate.In some embodiments, the liposomal composition comprises a liposomecomprising and α hexaglutamated Antifolate (Lp-αPANTIFOL). In furtherembodiments, the liposome comprises an L-α hexaglutamated Antifolate, aD-α hexaglutamated Antifolate, or an L- and D-α hexaglutamatedAntifolate. In some embodiments, the liposomal composition comprises aliposome that is anionic or neutral. In some embodiments, the liposomalcomposition comprises a liposome that is cationic. In some embodiments,the Lp-αPANTIFOL composition is not pegylated. In some embodiments, theLp-αPANTIFOL composition is non-targeted (NTLp-αPANTIFOL). In otherembodiments, the Lp-αPANTIFOL composition is targeted (TLp-αPANTIFOL).In some embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 20 nm to 500 nm, or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome having a diameter in the range of 20 nm to 400 nm, or any rangetherein between. In some embodiments, the liposomal compositioncomprises a liposome having a diameter in the range of 20 nm to 300 nm,or any range therein between. In some embodiments, the liposomalcomposition comprises a liposome having a diameter in the range of 20 nmto 200 nm, or any range therein between. In further embodiments, theliposomal composition comprises a liposome having a diameter in therange of 20 nm to 150 nm, or any range therein between. In furtherembodiments, the liposomal composition comprises a liposome having adiameter in the range of 80 nm to 120 nm, or any range therein between.In additional embodiments, 30-70%, 30-60%, or 30-50% w/w alphapolyglutamated Antifolate, or any range therein between, is encapsulated(entrapped) in the Lp-αPANTIFOL. In some embodiments, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% ormore than 75%, alpha polyglutamated Antifolate, is encapsulated in theLp-αPANTIFOL during the process of preparing the liposomes.

In some embodiments, the provided liposomes further comprise animmunostimulatory agent, a detectable marker, or both disposed on itsexterior. The immunostimulatory agent or detectable marker can beionically bonded or covalently bonded to an exterior of the liposome,including, for example, optionally to a steric stabilizer component ofthe liposome.

The terms “immunostimulatory agents”, also known as “immunostimulants”,and “immunostimulators”, refer to substances that stimulate an immune(including a preexisting immune response) by inducing activation orincreasing activity of any of the components of the immune system. Theseimmunostimulatory agents can include one or more of a hapten, anadjuvant, a protein immunostimulating agent, a nucleic acidimmunostimulating agent, and a chemical immunostimulating agent. Manyadjuvants contain a substance designed to stimulate immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A; IFN-gamma, IFN-alpha,FLT3-ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4,anti-CD28, anti-CD3). Cytokines, such as GM-CSF, interleukin-2, -7, -12,and -15, and other like growth factors, can also be used as adjuvants.In a preferred embodiment, the immunostimulant can be at least oneselected from fluorescein, DNP, beta glucan, beta-1,3-glucan,beta-1,6-glucan. In an additional preferred embodiment, theimmunostimulant is a Toll-like receptor (TLR) modulating agent. Infurther embodiments, the Toll-like receptor (TLR) modulating agent isone or more of: an oxidized low-density lipoprotein (e.g., OXPAC, PGPC),an eritoran lipid (e.g., E5564), and a resolvin. In some embodiments,the liposomes comprise fluorescein isothiocyanate (FITC) which, based onour experiments, surprisingly serves as both an immunostimulant and adetectable marker.

In some embodiments, the provided liposomes further comprise an agent,that increase uptake of liposomes into a cellular compartment ofinterest including the cytosol. In some embodiments, the agent providesthe liposome contents with the ability to bypass lysosomes (e.g.,chloroquine). In some embodiments, the agent improves the update of theliposome contents by mitochondria (e.g., sphingomyelin and a componentof mitoport).

In some embodiments, the liposomes comprise a detectable marker. Adetectable marker may, for example, include, at least, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator, an enzyme, a dye, an ink, a magneticcompound, a biocatalyst or a pigment that is detectable by any suitablemeans known in the art, e.g., magnetic resonance imaging (MRI), opticalimaging, fluorescent/luminescent imaging, or nuclear imaging techniques.

In some embodiments, the immunostimulatory agent and/or detectablemarker is attached to the exterior by co-incubating it with theliposome. For example, the immunostimulatory agent and/or detectablemarker may be associated with the liposomal membrane by hydrophobicinteractions or by an ionic bond such as an avidin/biotin bond or ametal chelation bond (e.g., Ni-NTA). Alternatively, theimmunostimulatory agent or detectable marker may be covalently bonded tothe exterior of the liposome such as, for example, by being covalentlybonded to a liposomal component or to the steric stabilizer which is thePEG.

One example reagent is fluorescein isothiocyanate (FITC) which, based onour experiments, surprisingly serves as both an immunostimulant and adetectable marker.

In some embodiments, the liposomes further comprise an agent thatincreases the uptake of liposomes into a cellular compartment ofinterest including the cytosol.

In some embodiments, the liposomes comprise a mitochondrial-targetingagent. In some embodiments, the liposomes comprise triphenylphosphonium(TPP). Methods and mechanisms for surface functionalizing liposomes withTPP are known in the art (e.g., attaching TPP to the lipid anchor via apeg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP)). In some embodiments, the liposomescomprise high-density octa-arginine. In some embodiments, the liposomescomprise sphingomyelin and/or a sphingomyelin metabolite. Sphingomyelinmetabolite used to formulate the liposomes of the present invention caninclude, for example ceramide, sphingosine or sphingosine 1-phosphate.In some embodiments, the liposomes comprise Rhodamine 123. In someembodiments, the liposomes comprise, a mitochondria penetrating peptide.In some embodiments, the liposomes comprise, a mitochondria penetratingagent selected from: a mitofusin peptide, a mitochondrial targetingsignal peptide, and Antennapedia helix III homeodomain cell-penetratingpeptide (ANT) (e.g., comprising RQIKIWFQNRRMKWKKRKKRRQR RR (SEQ IDNO:1), RKKRRXRRRGC where X is any natural or non-natural amino acid (SEQID NO:2), CCGCCAAGAAGCG (SEQ ID NO:3),GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO:4),ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCC GTCAGCTC (SEQ IDNO:5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAG AAGCGACCCCTCCGGGGCGAGCTG(SEQ ID NO:6)), or a mitochondrial penetrating fragment thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise a mitochondria penetrating agent selected from the group: aguanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium,monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, aproline-rich dendrimer, and a phosphonium salt (e.g.,methyltriphenyl-phosphonium and/or tetraphenylphosphonium).

In some embodiments, liposomes in the provided liposome compositionscomprise sphingomyelin and/or stearyl-octa-arginine. In someembodiments, the liposomes comprise sphingomyelin and/orstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine at a molar ratio of 9:2:1. In some embodiments,the liposomes comprise the MITO-porter® system or a variant thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise an agent such as a cell penetrating agent that that facilitatesdelivery of the liposome across a cell membrane and provides theliposome with the ability to bypass the endocytic pathway and the harshenvironment of lysosomes. Cell penetrating agents are known in the artand can routinely be used and adapted for manufacture and use of theprovided liposome compositions. In some embodiments, the cellpenetrating/lysosome bypassing agent is chloroquine. In someembodiments, the cell penetrating agent is a cell penetrating peptide.In some embodiments, liposomes in the provided liposome compositionscomprise a cell penetrating agent selected from the group: RKKRRQRRR(SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9),AAVAL LPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11), GALFLGFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13),RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKIWFQNRRMKWKK (SEQ ID NO:15),GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17),RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQID NO:20), LGIPAQEY (SEQ ID NO:21), LGIPEAEY (SEQ ID NO:22), LGIPEQAY(SEQ ID NO:23), LGIAEAEY (SEQ ID NO:24), LGIPEAAY (SEQ ID NO:25),LGIAEQAY (SEQ ID NO:26), LGIAEAAY (SEQ ID NO:27), LLIILRRRIRKQAHAHSK(SEQ ID NO:28), LKALAALAKKIL (SEQ ID NO:29), KLALKLALKALKAALKLA (SEQ IDNO:30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32),DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34),RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRRPPQ (SEQ ID NO:36),GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRR (SEQ ID NO:38),RRRRRRR (SEQ ID NO:39), RRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ IDNO:41), RRRRRRRR RR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43), andYTIWMPENPRPGT PCDIFTNSRGKRASNGGG G(R)n wherein n=2-15 R in the L- and/orD-form (SEQ ID NO:44), or a cell permeating fragment thereof.

As discussed above, the liposomes may comprise a steric stabilizer thatcan increase their longevity in circulation. For those embodiments,which incorporate a steric stabilizer, the steric stabilizer may be atleast one member selected from polyethylene glycol (PEG), poly-L-lysine(PLL), monosialoganglioside (GM1), poly(vinyl pyrrolidone) (PVP),poly(acrylamide) (PAA), poly(2-methyl-2-oxazoline),poly(2-ethyl-2-oxazoline), phosphatidyl polyglycerol,poly[N-(2-hydroxypropyl) methacrylamide], amphiphilicpoly-N-vinylpyrrolidones, L-amino-acid-based polymer, oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol. In some embodiments, the stericstabilizer or the population of steric stabilizers is PEG. In oneembodiment, the steric stabilizer is a PEG. In a further embodiment, thePEG has a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

In some embodiments, the disclosure provides liposomal compositions thatcomprise a pegylated liposome. In some embodiments, the pegylatedliposome comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the pegylated liposomecomprises a polyglutamated Antifolate described in Section II. In someembodiments, a pegylated liposome in the liposomal composition comprisesa αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the alpha polyglutamated Antifolate in theLp-αPANTIFOL comprises two or more glutamyl groups in the L-form. Inother embodiments, the alpha polyglutamated Antifolate in theLp-αPANTIFOL comprises a glutamyl group in the D-form. In furtherembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In additional embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL comprises two or more glutamyl groupsthat have a gamma linkage. In some embodiments, at least one glutamylgroup has both an alpha linkage and a gamma linkage. In someembodiments, the liposomal composition comprises a pegylated liposomethat comprises an α tetraglutamated Antifolate. In further embodiments,the liposome comprises an L-α tetraglutamated Antifolate, a D-αtetraglutamated Antifolate, or an L- and D-α tetraglutamated Antifolate.In some embodiments, the liposomal composition comprises a pegylatedliposome that comprises an a pentaglutamated Antifolate. In furtherembodiments, the liposome comprises an L-α pentaglutamated Antifolate, aD-α pentaglutamated Antifolate, or an L- and D-α pentaglutamatedAntifolate. In some embodiments, the liposomal composition comprises apegylated liposome that comprises a α hexaglutamated Antifolate. Infurther embodiments, the liposome comprises an L-α hexaglutamatedAntifolate, a D-α hexaglutamated Antifolate, or an L- and D-αhexaglutamated Antifolate. In some embodiments, the liposomalcomposition comprises a pegylated liposome according any of [25], and[27]-[69] of the Detailed Description. In some embodiments, theliposomal composition comprises a pegylated liposome that is anionic orneutral. In some embodiments, the liposomal composition comprises apegylated liposome that is cationic. In some embodiments, thePLp-αPANTIFOL composition is non-targeted (NTPLp-αPANTIFOL). In otherembodiments, the PLp-αPANTIFOL composition is targeted (TPLp-αPANTIFOL).In additional embodiments, the liposomal composition comprises apegylated liposome that comprises 30-70%, 30-60%, or 30-50% liposomeentrapped alpha polyglutamated Antifolate, or any range therein between.In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphapolyglutamated Antifolate is encapsulated (entrapped) in thePLp-αPANTIFOL. In some embodiments, the liposomal composition comprisesa pegylated liposome having a diameter in the range of 20 nm to 500 nm.In some embodiments, the liposomal composition comprises a pegylatedliposome having a diameter in the range of 20 nm to 200 nm. In furtherembodiments, the liposomal composition comprises a pegylated liposomehaving a diameter in the range of 80 nm to 120 nm.

In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80% or 90% ofthe polyglutamated Antifolate in the composition has 4-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, greater than 30%, 40%,50%, 60%, 70%, 80% or 90%, of the polyglutamated Antifolate in aprovided liposomal composition is tetraglutamated. In some embodiments,greater than 30%, 40%, 50%, 60%, 70%, 80% or 90%, of the polyglutamatedAntifolate in a provided liposomal composition is pentaglutamated. Insome embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80% or 90%, ofthe polyglutamated Antifolate in a provided liposomal composition ishexaglutamated.

In some embodiments, the alpha polyglutamated Antifolate compositions(e.g., polyglutamates and delivery vehicles such as liposomes containingthe polyglutamates) are in an aqueous solution. In some embodiments, thepolyglutamated Antifolate composition is administered in a liposomalcomposition at between about 0.005 and about 5000 mg/M² (meter of bodysurface area squared), or between about 0.1 and about 1000 mg/M², or anyrange therein between. In some embodiments, the αPANTIFOL composition isadministered in a liposomal composition at about 1 mg/kg to about 500mg/kg, 1 mg/kg to about 250 mg/kg, 1 mg/kg to about 200 mg/kg, 1 mg/kgto about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg/kg to about 20 mg/kg,about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, orabout 1 mg/kg to about 5 mg/kg, or any range therein between.

(1) Liposome Composition

The lipids and other components of the liposomes contained in theliposomal compositions can be any lipid, lipid combination and ratio, orcombination of lipids and other liposome components and their respectiveratios known in the art. However, it will be understood by one skilledin the art that liposomal encapsulation of any particular drug, such as,and without limitation, the alpha polyglutamated Antifolate discussedherein, may involve substantial routine experimentation to achieve auseful and functional liposomal formulation. In general, the providedliposomes may have any liposome structure, e.g., structures having aninner space sequestered from the outer medium by one or more lipidbilayers, or any microcapsule that has a semi-permeable membrane with alipophilic central part where the membrane sequesters an interior. Thelipid bilayer can be any arrangement of amphiphilic moleculescharacterized by a hydrophilic part (hydrophilic moiety) and ahydrophobic part (hydrophobic moiety). Usually amphiphilic molecules ina bilayer are arranged into two dimensional sheets in which hydrophobicmoieties are oriented inward the sheet while hydrophilic moieties areoriented outward. Amphiphilic molecules forming the provided liposomescan be any known or later discovered amphiphilic molecules, e.g., lipidsof synthetic or natural origin or biocompatible lipids. The liposomescan also be formed by amphiphilic polymers and surfactants, e.g.,polymerosomes and niosomes. For the purpose of this disclosure, withoutlimitation, these liposome-forming materials also are referred to as“lipids”.

The liposome composition formulations provided herein can be in liquidor dry form such as a dry powder or dry cake. The dry powder or dry cakemay have undergone primary drying under, for example, lyophilizationconditions or optionally, the dry cake or dry powder may have undergoneboth primary drying only or both primary drying and secondary drying. Inthe dry form, the powder or cake may, for example, have between 1% to 6%moisture, for example, such as between 2% to 5% moisture or between 2%to 4% moisture. One example method of drying is lyophilization (alsocalled freeze-drying, or cryodessication). Any of the compositions andmethods of the disclosure may include liposomes, lyophilized liposomesor liposomes reconstituted from lyophilized liposomes. In someembodiments, the disclosed compositions and methods include one or morelyoprotectants or cryoprotectants. These protectants are typicallypolyhydroxy compounds such as sugars (mono-, di-, and polysaccharides),polyalcohols, and their derivatives, glycerol, or polyethyleneglycol,trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, oraminoglycosides. In further embodiments, the lyoprotectants orcryoprotectants comprise up to 10% or up to 20% of a solution outsidethe liposome, inside the liposome, or both outside and inside theliposome.

In some embodiments, the liposomes include a steric stabilizer thatincreases their longevity in circulation. One or more steric stabilizerssuch as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid(monosialoganglioside (GM1)) or others occupies the space immediatelyadjacent to the liposome surface and excludes other macromolecules fromthis space. Consequently, access and binding of blood plasma opsonins tothe liposome surface are hindered, and thus interactions of macrophageswith such liposomes, or any other clearing mechanism, are inhibited andlongevity of the liposome in circulation is enhanced. In someembodiments, the steric stabilizer or the population of stericstabilizers is a PEG or a combination comprising PEG. In furtherembodiments, the steric stabilizer is a PEG or a combination comprisingPEG with a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

The diameter of the disclosed liposomes is not particularly limited. Insome embodiments, the liposomal composition comprises a liposome havinga diameter in the range of 20 nm to 500 nm, or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome that contains a αPANTIFOL according to any of [1]-[12] of theDetailed Description and has a diameter in the range of 20 nm to 500 nm.In some embodiments, the liposome is a liposome composition according toany of [13]-[72] of the Detailed Description and has a diameter in therange of 20 nm to 500 nm. In some embodiments, the liposomal compositioncomprises a liposome having a diameter in the range of 20 nm to 400 nm,or any range therein between. In some embodiments, the liposomalcomposition comprises a liposome that contains a αPANTIFOL according toany of [1]-[12] of the Detailed Description and has a diameter in therange of 20 nm to 400 nm. In some embodiments, the liposome is aliposome composition according to any of [13]-[72] of the DetailedDescription and has a diameter in the range of 20 nm to 400 nm. In someembodiments, the liposomal composition comprises a liposome having adiameter in the range of 20 nm to 300 nm, or any range therein between.In some embodiments, the liposomal composition comprises a liposome thatcontains a αPANTIFOL according to any of [13]-[72] of the DetailedDescription and has a diameter in the range of 20 nm to 300 nm. In someembodiments, the liposome is a liposome composition according to any of[13]-[72] of the Detailed Description and has a diameter in the range of20 nm to 300 nm.

In some embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 20 nm to 200 nm, or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome that contains a αPANTIFOL according to any of [1]-[12] of theDetailed Description and has a diameter in the range of 20 nm to 200 nm.In some embodiments, the liposome is a liposome composition according toany of [13]-[72] of the Detailed Description and has a diameter in therange of 20 nm to 200 nm.

In some embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 20 nm to 150 nm, or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome that contains a αPANTFOL according to any of [1]-[12] of theDetailed Description and has a diameter in the range of 20 nm to 150 nm.In some embodiments, the liposome is a liposome composition according toany of [13]-[72] of the Detailed Description and has a diameter in therange of 20 nm to 150 nm. In further embodiments, the liposomalcomposition comprises a liposome having a diameter in the range of 30 nmto 150 nm, or any range therein between. In some embodiments, theliposomal composition comprises a liposome that contains a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description and has adiameter in the range of 30 nm to 150 nm. In some embodiments, theliposome is a liposome composition according to any of [13]-[72] of theDetailed Description and has a diameter in the range of 30 nm to 150 nm,or any range therein between. In further embodiments, the liposomalcomposition comprises a liposome having a diameter in the range of 80 nmto 120 nm, or any range therein between. In some embodiments, theliposomal composition comprises a liposome that contains a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description and has adiameter in the range of 80 nm to 120 nm. In some embodiments, theliposome is a liposome composition according to any of [13]-[72] of theDetailed Description and has a diameter in the range of 80 nm to 120 nm.In further embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 40 nm to 70 nm, or any range thereinbetween. In some embodiments, liposomes comprise a αPANTIFOL accordingto any of [1]-[12] of the Detailed Description and have a diameter inthe range of 40 nm-70 nm. In some embodiments, the liposome is aliposome composition according to any of [13]-[72] of the DetailedDescription and has a diameter in the range of 40 nm-70 nm.

The properties of liposomes are influenced by the nature of lipids usedto make the liposomes. A wide variety of lipids have been used to makeliposomes. These include cationic, anionic and neutral lipids. In someembodiments, the liposomes comprising the alpha polyglutamatedAntifolate are anionic or neutral. In other embodiments, the providedliposomes are cationic. The determination of the charge (e.g., anionic,neutral or cationic) can routinely be determined by measuring the zetapotential of the liposome. The zeta potential of the liposome can bepositive, zero or negative. In some embodiments, the zeta potential ofthe liposome is less than or equal to zero. In some embodiments, thezeta potential of the liposome is in a range of 0 to −150 mV. In anotherembodiment, the zeta potential of the liposome is in the range of −30 to−50 mV.

In some embodiments, cationic lipids are used to make cationic liposomeswhich are commonly used as gene transfection agents. The positive chargeon cationic liposomes enables interaction with the negative charge oncell surfaces. Following binding of the cationic liposomes to the cell,the liposome is transported inside the cell through endocytosis.

In some preferred embodiments, a neutral to anionic liposome is used. Ina preferred embodiment, an anionic liposome is used. Using a mixture of,for example, neutral lipids such as HSPC and anionic lipids such asPEG-DSPE results in the formation of anionic liposomes which are lesslikely to non-specifically bind to normal cells. Specific binding totumor cells can be achieved by using a tumor targeting antibody such as,for example, a folate receptor antibody, including, for example, folatereceptor alpha antibody, folate receptor beta antibody and/or folatereceptor delta antibody.

As an example, at least one (or some) of the lipids is/are amphipathiclipids, defined as having a hydrophilic and a hydrophobic portions(typically a hydrophilic head and a hydrophobic tail). The hydrophobicportion typically orients into a hydrophobic phase (e.g., within thebilayer), while the hydrophilic portion typically orients toward theaqueous phase (e.g., outside the bilayer). The hydrophilic portion cancomprise polar or charged groups such as carbohydrates, phosphate,carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl and other likegroups. The hydrophobic portion can comprise apolar groups that includewithout limitation long chain saturated and unsaturated aliphatichydrocarbon groups and groups substituted by one or more aromatic,cyclo-aliphatic or heterocyclic group(s). Examples of amphipathiccompounds include, but are not limited to, phospholipids, aminolipidsand sphingolipids.

Typically, for example, the lipids are phospholipids. Phospholipidsinclude without limitation phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol,phosphatidylserine, and the like. It is to be understood that otherlipid membrane components, such as cholesterol, sphingomyelin, andcardiolipin, can be used.

The lipids comprising the liposomes provided herein can be anionic andneutral (including zwitterionic and polar) lipids including anionic andneutral phospholipids. Neutral lipids exist in an uncharged or neutralzwitterionic form at a selected pH. At physiological pH, such lipidsinclude, for example, dioleoylphosphatidylglycerol (DOPG),diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.Examples of zwitterionic lipids include without limitationdioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine(DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids arenegatively charged at physiological pH. These lipids include withoutlimitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dode-canoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein asnon-cationic lipids. Such lipids may contain phosphorus but they are notso limited. Examples of non-cationic lipids include lecithin,lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine,dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanol-amine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidy 1-ethan-olamine (DSPE),palmitoyloleoyl-phosphatidylethanolamine (POPE)palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE,16-0-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), phosphatidylserine,phosphatidyl-inositol, sphingomyelin, cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetyl-phosphate, and cholesterol.

The liposomes may be assembled using any liposomal assembly method usingliposomal components (also referred to as liposome components) known inthe art. Liposomal components include, for example, lipids such as DSPE,HSPC, cholesterol and derivatives of these components. Other suitablelipids are commercially available for example, by Avanti Polar Lipids,Inc. (Alabaster, Ala., USA). A partial listing of available negativelyor neutrally charged lipids suitable for making anionic liposomes, canbe, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC,DOPC, DMPE, DPPE, DOPE, DMPA.Na, DPPA.Na, DOPA.Na, DMPG.Na, DPPG.Na,DOPG.Na, DMPS.Na, DPPS.Na, DOPS.Na, DOPE-Glutaryl.(Na)2, TetramyristoylCardiolipin .(Na)2, DSPE-mPEG-2000.Na, DSPE-mPEG-5000.Na, andDSPE-Maleimide PEG-2000.Na.

In some embodiments, the αPANTIFOL compositions provided herein areformulated in a liposome comprising a cationic lipid. In one embodiment,the cationic lipid is selected from, but not limited to, a cationiclipid described in Intl. Appl. Publ. Nos. WO2012/040184, WO2011/153120,WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460,WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865and WO2008/103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and 8,283,333 andUS Appl. Publ. Nos. US20100036115 and US20120202871; each of which isherein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid may be selected from, but not limited to,Formula A described in Intl. Appl. Publ. Nos. WO2012/040184,WO2011/153120, WO201/1149733, WO2011/090965, WO2011/043913,WO2011/022460, WO2012/061259, WO2012/054365 and WO2012/044638; each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, Formula CLI-CLXXIX of International Publication No.WO2008103276, Formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, FormulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and Formula I-VI of US PatentPublication No. US20100036115; each of which is herein incorporated byreference in their entirety. As a non-limiting example, the cationiclipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemyl-hexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine, (15Z, 18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyl-tricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexa-cosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyl-triaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethyl-heptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpenta-cos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclo-propyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethyl nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] nonadecan-10-amine,N,N-dimethyl-21-[(R1S,2R)-2-octylcyclopropyl] henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,—N,N-dimethyl-[(1R,2S)-2-undecyl-cyclopropyl] tetradecan-5-amine,—N,N-dimethyl-3-{7-[(1S, 2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-penta-decan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-n-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl] ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-xy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy) propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-pan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-an-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl1-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dime-thyl-propan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octa-deca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-die-n-1-yloxylpropan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-1{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl} oxy) propan-2-amine,N,N-dimethyl-1-{[-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or acid or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in in Intl. Publ. No. WO2012/170889, which is hereinincorporated by reference in its entirety

The cationic lipid can routinely be synthesized using methods known inthe art and/or as described in Intl. Publ. Nos. WO2012/040184,WO2011/153120, WO2011/149733, WO2011/090965, WO201/1043913,WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638,WO2010/080724 and WO2010/21865; each of which is herein incorporated byreference in its entirety.

Lipid derivatives can include, for example, at least, the bonding(preferably covalent bonding) of one or more steric stabilizers and/orfunctional groups to the liposomal component after which the stericstabilizers and/or functional groups should be considered part of theliposomal components. Functional groups comprises groups that can beused to attach a liposomal component to another moiety such as aprotein. Such functional groups include, at least, maleimide. Thesesteric stabilizers include at least one from polyethylene glycol (PEG);poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone)(PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;poly[N-(2-hydroxy-propyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinylalcohol.

In some embodiments, the αPANTIFOL compositions are formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished using methods known in the art and/or as describedin U.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012/013326; herein incorporatedby reference in its entirety. In another embodiment, the αPANTIFOL isformulated in a lipid-polycation complex which further includes aneutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

Since the components of a liposome can include any molecule(s) (i.e.,chemical/reagent/protein) that is bound to it, in some embodiments, thecomponents of the provided liposomes include, at least, a memberselected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG;HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide.In some embodiments, the components of the provided liposomes includeDSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide;cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferredembodiment, the liposomal components that make up the liposome comprisesDSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

In additional embodiments, the liposomes of the liposome compositionsprovided herein comprise oxidized phospholipids. In some embodiments,the liposomes comprise an oxidize phospholipid of a member selected fromphosphatidylserines, phosphatidylinositols, phosphatidylethanolamines,phosphatidylcholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments,the phospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Theterm “oxPAPC”, as used herein, refers to lipids generated by theoxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine(PAPC), which results in a mixture of oxidized phospholipids containingeither fragmented or full length oxygenated sn-2 residues.Well-characterized oxidatively fragmented species contain a five-carbonsn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxidationof arachidonic acid residue also produces phospholipids containingesterified isoprostanes. In some embodiments, the oxPAPC includesHOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species, among other oxidizedproducts present in oxPAPC. In further embodiments, the oxPAPCs areepoxyisoprostane-containing phospholipids. In further embodiments, theoxPAPC is 1-palmitoyl-2-(5,6-epoxyisoprostaneE2)-sn-glycero-3-phosphocholine (5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, thephospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomal alpha polyglutamated Antifolatecomposition is pegylated (i.e., a pegylated liposomal alphapolyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate(PLp-αPANTIFOL or PLp-αPANTIFOL). In some embodiments, the PLp-αPANTIFOLor PLp-αPANTIFOL is water soluble. That is, the PLp-αPANTIFOL orPLp-αPANTIFOL is in the form an aqueous solution.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise a lipid selected from:1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC);1-palmitoyl-2-(9′oxo-nonanoyl)-sn-glycero-3-phosphocholine;1-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and1-palmitoyl-2-acetoyl-sn-glycero-3-phospho-choline. In furtherembodiments, the liposome comprises PGPC.

In some embodiments, the pH of solutions comprising the liposomecomposition is from pH 2 to 8, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom pH 5 to 8 or from pH 2 to 6, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom pH 5 to 8, or any range therein between. In some embodiments, thepH of solutions comprising the liposome composition is from pH 6 to 7,or any range therein between. In some embodiments, the pH of solutionscomprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5,from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.

In some embodiments, at least one component of the liposome lipidbilayer is functionalized (or reactive). As used herein, afunctionalized component is a component that comprises a reactive groupthat can be used to crosslink reagents and moieties to the lipid. If thelipid is functionalized, any liposome that it forms is alsofunctionalized. In some embodiments, the reactive group is one that willreact with a crosslinker (or other moiety) to form crosslinks. Thereactive group in the liposome lipid bilayer is located anywhere on thelipid that allows it to contact a crosslinker and be crosslinked toanother moiety (e.g., a steric stabilizer or targeting moiety). In someembodiments, the reactive group is in the head group of the lipid,including for example a phospholipid. In some embodiments, the reactivegroup is a maleimide group. Maleimide groups can be crosslinked to eachother in the presence of dithiol crosslinkers including but not limitedto dithiolthrietol (DTT).

It is to be understood that the use of other functionalized lipids,other reactive groups, and other crosslinkers beyond those describedabove is further contemplated. In addition to the maleimide groups,other examples of contemplated reactive groups include but are notlimited to other thiol reactive groups, amino groups such as primary andsecondary amines, carboxyl groups, hydroxyl groups, aldehyde groups,alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydrylgroups, and pyridyl disulfide groups.

Functionalized and non-functionalized lipids are available from a numberof commercial sources including Avanti Polar Lipids (Alabaster, Ala.)and Lipoid LLC (Newark, N.J.).

(2) Liposome Interior Space

In further non-limiting embodiments, the provided liposomes enclose aninterior space. In some embodiments, the interior space comprises, butis not limited to, an aqueous solution. In some embodiments, theinterior space comprises an alpha polyglutamated Antifolate as providedherein. In additional embodiments, the interior space of the liposomecomprises a tonicity agent. In some embodiments. In some embodiments,the concentration (weight percent) of the tonicity agent is 0.1-20%,1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therein between. In someembodiments, the interior space of the liposome includes a sugar (e.g.,trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol,dextrose, fructose, etc.). In further embodiments, the concentration(weight percent) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, or1-50%, or any range therein between. In some embodiments, the pH of theinterior space of the liposome is from pH 2 to 8, or any range thereinbetween. In some embodiments, the pH of solutions comprising theliposome composition is from pH 5 to 8, or any range therein between. Insome embodiments, the pH of solutions comprising the liposomecomposition is from pH 6 to 7, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between. In some embodiments, the interior spacecomprises buffer. In further embodiments, the buffer a buffer selectedfrom HEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasicsodium phosphate). In some embodiments, the buffer is HEPES. In someembodiments, the buffer is citrate. In some embodiments, the buffer issodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). Insome embodiments, the buffer is at a concentration of 15 to 200 mM, orany range therein between. In further embodiments, the buffer is at aconcentration of between 5 to 200 mM, 15-200, between 5 to 100 mM,between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range thereinbetween. In some embodiments, the buffer is HEPES at a concentration of15 to 200 mM, or any range therein between. In some embodiments, thebuffer is citrate at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is sodium phosphate ata concentration of 15 to 200 mM, or any range therein between. In someembodiments, the interior space of the liposome comprises a totalconcentration of sodium acetate and calcium acetate of between 5 mM to500 mM, or 50 mM to 500 mM, or any range therein between.

In some embodiments, the interior space of the liposome includesglutamine, glutamate, and/or polyglutamate (e.g., diglutamate,triglutamate, tetraglutamate, and/or pentaglutamate, containing one ormore gamma glutamyl group linkages or 1 or more alpha glutamyllinkages). In further embodiments, the concentration weight percent ofthe glutamine, glutamate, and/or polyglutamate is 0.1-20%, 1-20%,0.5-15%, 1%-15%, 5-20%, or 1-50%, or any range therein between. In someembodiments the interior space of the liposome includes glutamine. Insome embodiments the interior space of the liposome includes glutamate.In some embodiments the interior space of the liposome includespolyglutamate. In some embodiments, the concentration (weight percent)of glutamine, glutamate, and/or polyglutamate is 1-15%, or any rangetherein between. In an additional embodiment, the glutamine, glutamate,and/or polyglutamate is present at about 5% to 20% weight percent oftrehalose or any combination of one or more lyoprotectants orcryoprotectants at a total concentration of 5% to 20%. In someembodiments, the interior space comprises buffer. In furtherembodiments, the buffer is HEPES buffer or citrate buffer. In furtherembodiments, the citrate buffer is at a concentration of between 5 to200 mM. In some embodiments, the interior space has a pH of between 2.8to 6. In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between. In some embodiments, theinterior space of the liposome comprises a total concentration of sodiumacetate and calcium acetate of between 50 mM to 500 mM.

In some embodiments, the interior space of the liposome includesglutamine. In further embodiments, the concentration weight percent ofthe glutamine is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, 5-20%, or 1-50%, orany range therein between. In some embodiments, the concentration(weight percent) of glutamine is 1-15%, or any range therein between. Inan additional embodiment, the glutamine is present at about 5% to 20%weight percent of glutamine or any combination of one or morelyoprotectants or cryoprotectants at a total concentration of 5% to 20%.In some embodiments, the interior space comprises buffer. In furtherembodiments, the buffer is HEPES buffer or citrate buffer. In furtherembodiments, the citrate buffer is at a concentration of between 5 to200 mM. In some embodiments, the interior space has a pH of between 2.8to 6. In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between. In some embodiments, theinterior space of the liposome comprises a total concentration of sodiumacetate and calcium acetate of between 50 mM to 500 mM.

In some embodiments, the interior space of the liposome includestrehalose. In further embodiments, the concentration weight percent oftrehalose is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, 5-20%, or 1-50%, or anyrange therein between. In further embodiments, the concentration (weightpercent) of trehalose is 1-15%, or any range therein between. In anadditional embodiment, the trehalose is present at about 5% to 20%weight percent of trehalose or any combination of one or morelyoprotectants or cryoprotectants at a total concentration of 5% to 20%.In some embodiments, the interior space comprises buffer. In furtherembodiments, the buffer is HEPES buffer or citrate buffer. In furtherembodiments, the citrate buffer is at a concentration of between 5 to200 mM. In some embodiments, the interior space has a pH of between 2.8to 6. In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between. In some embodiments, theinterior space of the liposome comprises a total concentration of sodiumacetate and calcium acetate of between 50 mM to 500 mM.

In some embodiments, the interior space of the liposome includesdextrose. In further embodiments, the concentration weight percent ofdextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, 5-20%, or 1-50%, or anyrange therein between. In yet further embodiments, the concentration(weight percent) of dextrose is 1-15%, or any range therein between. Inan additional embodiment, the dextrose is present at about 5% to 20%weight percent of dextrose or any combination of one or morelyoprotectants or cryoprotectants at a total concentration of 5% to 20%.In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In yet further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between.

In additional embodiments, the disclosure provides liposomalcompositions that comprise a liposome encapsulating (i.e., filled with)an alpha polyglutamated Antifolate e.g., a αPANTIFOL disclosed herein).In some embodiments, the liposomal composition comprises a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the liposomal composition comprises a polyglutamatedAntifolate described in Section II. In some embodiments, a liposome inthe liposomal composition comprises an ANTIFOL containing 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups (including the glutamyl group ofthe Antifolate). In some embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL comprises two or more glutamyl groups inthe L-form. In other embodiments, the alpha polyglutamated Antifolate inthe Lp-αPANTIFOL comprises a glutamyl group in the D-form. In furtherembodiments, the alpha polyglutamated Antifolate in the Lp-αPANTIFOLcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In additional embodiments, the alpha polyglutamatedAntifolate in the Lp-αPANTIFOL comprises two or more glutamyl groupsthat have a gamma carboxyl linkage. In some embodiments, the liposomalcomposition comprises a liposome comprising a α pentaglutamatedAntifolate. In further embodiments, the liposome comprises an L-αpentaglutamated Antifolate, a D-α pentaglutamated Antifolate, or an L-and D-α pentaglutamated Antifolate. In some embodiments, the liposomalcomposition comprises a liposome comprising a α hexaglutamatedAntifolate (Lp-αPANTIFOL). In further embodiments, the liposomecomprises an L-α hexaglutamated Antifolate, a D-α hexaglutamatedAntifolate, or an L- and D-α hexaglutamated Antifolate. In someembodiments, the liposomal composition is a liposomal compositionaccording to any of [13]-[72] of the Detailed Description.

In some embodiments, the disclosure provides a liposomal compositioncomprising a targeted and pegylated liposome that comprises an alphapolyglutamated Antifolate (TPLp-αPANTIFOL). In some embodiments, theliposomal composition comprises a αPANTIFOL according to any of [1]-[12]of the Detailed Description. In some embodiments, the liposomalcomposition comprises a polyglutamated Antifolate described in SectionII. In some embodiments, the liposomal composition is a targetedpegylated liposomal composition according to any of [55]-[74] of theDetailed Description. In some embodiments, the targeted pegylatedliposomal alpha polyglutamated (e.g., pentaglutamated or hexaglutamated)Antifolate comprises a medium comprising a liposome including aninterior space; an aqueous alpha polyglutamated Antifolate disposedwithin the interior space; and a targeting moiety comprising a proteinwith specific affinity for at least one folate receptor, and wherein thetargeting moiety disposed at the exterior of the liposome. In someembodiments, the medium is an aqueous solution. In some embodiments, theinterior space, the exterior space (e.g., the medium), or both theinterior space and the medium contains one or more lyoprotectants orcryoprotectants which are listed above. In some embodiments, thecryoprotectant is mannitol, trehalose, sorbitol, or sucrose.

In some embodiments, the liposome encapsulating alpha polyglutamatedAntifolate (e.g., Lp-αPANTIFOL, including PLp-αPANTIFOL, TPLp-αPANTIFOL,TLp-αPANTIFOL, and NTLp-αPANTIFOL) has an interior space that containsless than 500,000 or less than 200,000 molecules of alpha polyglutamatedAntifolate. In some embodiments, the liposome interior space containsbetween 10 to 100,000 molecules of alpha polyglutamated Antifolate, orany range therein between. In some embodiments, the liposome interiorspace contains between 10,000 to 100,000 molecules of alphapolyglutamated Antifolate, or any range therein between. In someembodiments, the liposome is not pegylated and has an interior spacethat contains less than 500,000 or less than 200,000 molecules of alphapolyglutamated Antifolate. In some embodiments, the liposome is notpegylated and the interior space of the liposome contains between 10 to100,000 molecules of alpha polyglutamated Antifolate, or any rangetherein between. In further embodiments, the liposome is not pegylatedand the interior space of the liposome contains between 10,000 to100,000 molecules of alpha polyglutamated Antifolate, or any rangetherein between. In some embodiments, the liposome comprises a targetingmoiety, is not pegylated (TLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphapolyglutamated Antifolate. In some embodiments, the liposome comprises atargeting moiety, is not pegylated, and the interior space of theliposome contains between 10 to 100,000 molecules of alphapolyglutamated Antifolate, or any range therein between. In furtherembodiments, the liposome comprises a targeting moiety, is notpegylated, and the interior space of the liposome contains between10,000 to 100,000 molecules of alpha polyglutamated Antifolate, or anyrange therein between. In some embodiments, the liposome does notcomprise a targeting moiety, is not pegylated, (NTLp-αPANTIFOL) and hasan interior space that contains less than 500,000 or less than 200,000molecules of alpha polyglutamated Antifolate. In some embodiments, theliposome does not comprise a targeting moiety, is not pegylated, and theinterior space of the liposome contains between 10 to 100,000 moleculesof alpha polyglutamated Antifolate, or any range therein between. Infurther embodiments, the liposome does not comprise a targeting moiety,is not pegylated, and the interior space of the liposome containsbetween 10,000 to 100,000 molecules of alpha polyglutamated Antifolate,or any range therein between.

In some embodiments, the liposome encapsulates an alpha polyglutamatedAntifolate that contains 2-10 glutamyl groups (i.e., Lp-αPANTIFOL,including PLp-αPANTIFOL, TPLp-αPANTIFOL, TLp-αPANTIFOL, andNTLp-αPANTIFOL) and has an interior space that contains less than500,000 or less than 200,000 molecules of alpha polyglutamatedAntifolate containing 2-10 glutamyl groups. In some embodiments, theliposome interior space contains between 10 to 100,000 molecules ofalpha polyglutamated Antifolate containing 2-10 glutamyl groups, or anyrange therein between. In further embodiments, the liposome interiorspace contains between 10,000 to 100,000 molecules of alphapolyglutamated Antifolate containing 2-10 glutamyl groups, or any rangetherein between. In some embodiments, the liposome is not pegylated andhas an interior space that contains less than 500,000 or less than200,000 molecules of alpha polyglutamated Antifolate containing 2-10glutamyl groups. In some embodiments, the liposome is a liposomalcomposition according to any of [13]-[72] of the Detailed Description.In some embodiments, the liposome is not pegylated and the interiorspace of the liposome contains between 10 to 100,000 molecules of alphapolyglutamated Antifolate containing 2-10 glutamyl groups, or any rangetherein between. In further embodiments, the liposome is not pegylatedand the interior space of the liposome contains between 10,000 to100,000 molecules of alpha polyglutamated Antifolate containing 2-10glutamyl groups, or any range therein between. In some embodiments, theliposome comprises a targeting moiety, is not pegylated (TLp-αPANTIFOL)and has an interior space that contains less than 500,000 or less than200,000 molecules of alpha polyglutamated Antifolate containing 2-10glutamyl groups. In some embodiments, the liposome comprises a targetingmoiety, is not pegylated, and the interior space of the liposomecontains between 10 to 100,000 molecules of alpha polyglutamatedAntifolate containing 2-10 glutamyl groups, or any range thereinbetween. In further embodiments, the liposome comprises a targetingmoiety, is not pegylated, and the interior space of the liposomecontains between 10,000 to 100,000 molecules of alpha polyglutamatedAntifolate containing 2-10 glutamyl groups, or any range thereinbetween. In some embodiments, the liposome is non-targeted andunpegylated (αPANTIFOL) and has an interior space that contains lessthan 500,000 or less than 200,000 molecules of alpha polyglutamatedAntifolate containing 2-10 glutamyl groups. In some embodiments, theliposome does not comprise a targeting moiety, is not pegylated, and theinterior space of the liposome contains between 10 to 100,000 moleculesof alpha polyglutamated Antifolate containing 2-10 glutamyl groups, orany range therein between. In further embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10,000 to 100,000 molecules of alpha polyglutamatedAntifolate containing 2-10 glutamyl groups, or any range thereinbetween.

In some embodiments, the liposome encapsulates alpha tetraglutamatedAntifolate (i.e., Lp-αPANTIFOL, including PLp-αPANTIFOL, TPLp-αPANTIFOL,TLp-αPANTIFOL, and NTLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphatetraglutamated Antifolate. In some embodiments, the liposome interiorspace contains between 10 to 100,000 molecules of alpha tetraglutamatedAntifolate, or any range therein between. In some embodiments, theliposome interior space contains between 10,000 to 100,000 molecules ofalpha tetraglutamated Antifolate, or any range therein between. In someembodiments, the liposome is not pegylated and has an interior spacethat contains less than 500,000 or less than 200,000 molecules of alphatetraglutamated Antifolate. In some embodiments, the liposome is notpegylated and the interior space of the liposome contains between 10 to100,000 molecules of alpha tetraglutamated Antifolate, or any rangetherein between. In further embodiments, the liposome is not pegylatedand the interior space of the liposome contains between 10,000 to100,000 molecules of alpha tetraglutamated Antifolate, or any rangetherein between. In some embodiments, the liposome comprises a targetingmoiety, is not pegylated (TLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphatetraglutamated Antifolate. In some embodiments, the liposome comprisesa targeting moiety, is not pegylated, and the interior space of theliposome contains between 10 to 100,000 molecules of alphatetraglutamated Antifolate, or any range therein between. In furtherembodiments, the liposome comprises a targeting moiety, is notpegylated, and the interior space of the liposome contains between10,000 to 100,000 molecules of alpha tetraglutamated Antifolate, or anyrange therein between. In some embodiments, the liposome is non-targetedand unpegylated (NTLp-αPANTIFOL) and has an interior space that containsless than 500,000 or less than 200,000 molecules of alphatetraglutamated Antifolate. In some embodiments, the liposome does notcontain a targeting moiety and is not pegylated, and the interior spaceof the liposome contains between 10 to 100,000 molecules of alphatetraglutamated Antifolate, or any range therein between. In furtherembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10,000 to 100,000molecules of alpha tetraglutamated Antifolate, or any range thereinbetween.

In some embodiments, the liposome encapsulates alpha pentaglutamatedAntifolate (i.e., Lp-αPANTIFOL, including PLp-αPANTIFOL, TPLp-αPANTIFOL,TLp-αPANTIFOL, and NTLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphapentaglutamated Antifolate. In some embodiments, the liposome interiorspace contains between 10 to 100,000 molecules of alpha pentaglutamatedAntifolate, or any range therein between. In some embodiments, theliposome interior space contains between 10,000 to 100,000 molecules ofalpha pentaglutamated Antifolate, or any range therein between. In someembodiments, the liposome is not pegylated and has an interior spacethat contains less than 500,000 or less than 200,000 molecules of alphapentaglutamated Antifolate. In some embodiments, the liposome is notpegylated and the interior space of the liposome contains between 10 to100,000 molecules of alpha pentaglutamated Antifolate, or any rangetherein between. In further embodiments, the liposome is not pegylatedand the interior space of the liposome contains between 10,000 to100,000 molecules of alpha pentaglutamated Antifolate, or any rangetherein between. In some embodiments, the liposome comprises a targetingmoiety, is not pegylated (TLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphapentaglutamated Antifolate. In some embodiments, the liposome comprisesa targeting moiety, is not pegylated, and the interior space of theliposome contains between 10 to 100,000 molecules of alphapentaglutamated Antifolate, or any range therein between. In furtherembodiments, the liposome comprises a targeting moiety, is notpegylated, and the interior space of the liposome contains between10,000 to 100,000 molecules of alpha pentaglutamated Antifolate, or anyrange therein between. In some embodiments, the liposome is non-targetedand unpegylated (NTLp-αPANTIFOL) and has an interior space that containsless than 500,000 or less than 200,000 molecules of alphapentaglutamated Antifolate. In some embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10 to 100,000 molecules of alpha pentaglutamatedAntifolate, or any range therein between. In further embodiments, theliposome is non-targeted and unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of alphapentaglutamated Antifolate, or any range therein between.

In some embodiments, the liposome encapsulates alpha hexaglutamatedAntifolate (i.e., Lp-αPANTIFOL, including PLp-αPANTIFOL, TPLp-αPANTIFOL,TLp-αPANTIFOL, and NTLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphahexaglutamated Antifolate. In some embodiments, the liposome interiorspace contains between 10 to 100,000 molecules of alpha hexaglutamatedAntifolate, or any range therein between. In further embodiments, theliposome interior space contains between 10,000 to 100,000 molecules ofalpha hexaglutamated Antifolate, or any range therein between. In someembodiments, the liposome is not pegylated and has an interior spacethat contains less than 500,000 or less than 200,000 molecules of alphahexaglutamated Antifolate. In some embodiments, the liposome is notpegylated and the interior space of the liposome contains between 10 to100,000 molecules of alpha hexaglutamated Antifolate, or any rangetherein between. In further embodiments, the liposome is not pegylatedand the interior space of the liposome contains between 10,000 to100,000 molecules of alpha hexaglutamated Antifolate, or any rangetherein between. In some embodiments, the liposome comprises a targetingmoiety, is not pegylated (TLp-αPANTIFOL) and has an interior space thatcontains less than 500,000 or less than 200,000 molecules of alphahexaglutamated Antifolate. In some embodiments, the liposome comprises atargeting moiety, is not pegylated, and the interior space of theliposome contains between 10 to 100,000 molecules of alphahexaglutamated Antifolate, or any range therein between. In furtherembodiments, the liposome comprises a targeting moiety, is notpegylated, and the interior space of the liposome contains between10,000 to 100,000 molecules of alpha hexaglutamated Antifolate, or anyrange therein between. In some embodiments, the liposome is non-targetedand unpegylated (NTLp-αPANTIFOL) and has an interior space that containsless than 500,000 or less than 200,000 molecules of alpha hexaglutamatedAntifolate. In some embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of alpha hexaglutamated Antifolate, or any rangetherein between. In further embodiments, the liposome is non-targetedand unpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of alpha hexaglutamated Antifolate, or anyrange therein between.

In some embodiments, the disclosure provides a liposomal alphapolyglutamated Antifolate composition wherein the liposome encapsulatesalpha polyglutamated Antifolate or a salt or acid thereof, and one ormore aqueous pharmaceutically acceptable carriers. In some embodiments,the liposomal alpha polyglutamated Antifolate composition is a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the liposome comprises a polyglutamated Antifolatedescribed in Section II. In some embodiments, the liposome interiorspace contains trehalose. In some embodiments, the liposome interiorspace contains 1% to 50% weight of trehalose. In some embodiments, theliposome interior space contains HBS at a concentration of between 1 to200 mM and a pH of between 2 to 8. In some embodiments, liposomeinterior space has a pH 5-8, or any range therein between. In someembodiments, liposome interior space has a pH 6-7, or any range thereinbetween. In some embodiments, the liposome interior space has a totalconcentration of sodium acetate and calcium acetate of between 50 mM to500 mM, or any range therein between.

A Non-Polyglutamated Polyglutamatable Antifolates

In some embodiments, the liposome alpha polyglutamated Antifolate (i.e.,Lp-αPANTIFOL, including PLp-αPANTIFOL, TPLp-αPANTIFOL, TLp-αPANTIFOL,and NTLp-αPANTIFOL) compositions comprise alpha polyglutamatedAntifolate (e.g., an αPANTIFOL disclosed 2] of the Detailed Descriptionherein) and one or more non-polyglutamated, polyglutamatable antifolatecompositions.

In some embodiments, the Lp-αPANTIFOL (e.g., PLp-αPANTIFOL,TPLp-αPANTIFOL, TLp-αPANTIFOL, and NTLp-αPANTIFOL) comprises an alphapolyglutamated Antifolate (e.g., a αPANTIFOL disclosed herein) and theAntifolate (ANTIFOL). In some embodiments, the Lp-αPANTIFOL comprises aαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the liposome comprises a polyglutamated Antifolatedescribed in Section II. In some embodiments, the Lp-αPANTIFOL is aliposomal composition according to any of [13]-[72] of the DetailedDescription.

In some embodiments, the Lp-αPANTIFOL (i.e., liposome alphapolyglutamated Antifolate) comprises alpha polyglutamated Antifolate anda polyglutamatable antifolate selected from the group consisting of:methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed(RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In someembodiments, the Lp-αPANTIFOL comprises alpha polyglutamated Antifolateand lometrexol. In some embodiments, the Lp-αPANTIFOL comprises alphapolyglutamated Antifolate and pemetrexed. In some embodiments, theLp-αPANTIFOL comprises alpha polyglutamated Antifolate and leucovorin.In some embodiments, the Lp-αPANTIFOL comprises alpha polyglutamatedAntifolate and a triazine antifolate derivative (e.g., a sulphonylfluoride triazine such as NSC 127755). In some embodiments, theLp-αPANTIFOL comprises alpha polyglutamated Antifolate and a serinehydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, theSHMT2 inhibitor is an antifolate (e.g., a polyglutamatable ornonpolyglutamatable antifolate). In some embodiments, the SHMT2inhibitor is an antifolate.

B Non-Polyglutamatable Antifolates

In some embodiments, the Lp-αPANTIFOL (e.g., PLp-αPANTIFOL,TPLp-αPANTIFOL, TLp-αPANTIFOL, and NTLp-αPANTIFOL) comprises an alphapolyglutamated Antifolate (e.g., a αPANTIFOL disclosed herein) and aso-called “non-polyglutamatable” antifolate. In some embodiments, theliposome comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome comprises apolyglutamated Antifolate described in Section II. In some embodiments,the liposome comprises an alpha polyglutamated Antifolate and anon-polyglutamatable antifolate that inhibits one or more enzymes in thefolate cycle metabolic pathway. In further embodiments, thenon-polyglutamatable antifolate inhibits one or more enzymes selectedfrom: thymidylate synthase (TS), dihydrofolate reductase (DHFR),glycinamide ribonucleotide (GAR) transformylase, and aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. In some embodiments,the liposome comprises an alpha polyglutamated Antifolate and anon-polyglutamatable antifolate that inhibits DHFR. In some embodiments,the liposome comprises an alpha polyglutamated Antifolate and anon-polyglutamatable antifolate that inhibits TS. In some embodiments,the liposome comprises an alpha polyglutamated Antifolate and anon-polyglutamatable antifolate that inhibits GAR or AICARtransformylase. In further embodiments, the non-polyglutamatableantifolate is selected from: timetrexate (TMQ), piritrexim (BW301U), andtalotrexin (PT523). In further embodiments, the non-polyglutamatableantifolate is selected from: nolatrexed (AG337), plevitrexed (ZD9331,BGC9331), and BGC 945 (ONX 0801) or a pharmaceutically acceptable saltthereof.

C Platinums

In some embodiments, the liposome comprises an alpha polyglutamatedAntifolate (Lp-αPANTIFOL, such as e.g., PLp-αPANTIFOL, TPLp-αPANTIFOL,TLp-αPANTIFOL, and NTLp-αPANTIFOL) and a platinum-based chemotherapeuticagent or a salt or acid, thereof. In some embodiments, the liposomecontains a complex of an alpha polyglutamated Antifolate (e.g., aαPANTIFOL disclosed and a platinum based agent. In some embodiments, thealpha polyglutamated Antifolate/platinum-based agent complex comprises aαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the complex comprises a polyglutamated Antifolatedescribed in Section II.

In some embodiments, the Lp-αPANTIFOL comprises a platinum-basedchemotherapeutic agent selected from: cisplatin, carboplatin, andoxaliplatin, or a salt or acid thereof. In some embodiments, theliposome contains a complex of an alpha polyglutamated Antifolate and aplatinum-based chemotherapeutic agent selected from: cisplatin,carboplatin, and oxaliplatin, or a salt or acid thereof. In someembodiments, the Lp-αPANTIFOL comprises a αPANTIFOL according to any of[1]-[12] of the Detailed Description and a platinum-basedchemotherapeutic agent selected from: cisplatin, carboplatin, andoxaliplatin, or a salt or acid thereof. In some embodiments, theLp-αPANTIFOL comprises a complex of an alpha polyglutamated Antifolatecomplex comprising a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the complex comprises apolyglutamated Antifolate described in Section II. In other embodiments,the Lp-αPANTIFOL comprises an analog of a platinum-basedchemotherapeutic agent selected from: cisplatin, carboplatin, oroxaliplatin, or a salt or acid thereof.

In some embodiments, the Lp-αPANTIFOL comprises an alpha polyglutamatedAntifolate and cisplatin or a salt or acid thereof. In some embodiments,the Lp-αPANTIFOL comprises an alpha polyglutamated Antifolate and acisplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-αPANTIFOL comprises an alpha polyglutamatedAntifolate and carboplatin, or a salt or acid thereof. In someembodiments, the liposome comprises an alpha polyglutamated Antifolateand carboplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-αPANTIFOL comprises an alpha polyglutamatedAntifolate and oxaliplatin, or a salt or acid thereof. In someembodiments, the liposome comprises an alpha polyglutamated Antifolateand an oxaliplatin analog, or a salt or acid thereof.

In some embodiments, the liposome comprises an alpha polyglutamatedAntifolate (e.g., a αPANTIFOL disclosed herein) and a platinum-basedchemotherapeutic agent selected from: nedaplatin, heptaplatin, andlobaplatin, nedaplatin, heptaplatin, and lobaplatin or a salt or acidthereof. In some embodiments, the Lp-αPANTIFOL comprises an alphapolyglutamated Antifolate and an analog of a platinum-basedchemotherapeutic agent selected from: nedaplatin, heptaplatin, andlobaplatin, or a salt or acid thereof.

In some embodiments, the Lp-αPANTIFOL comprises an alpha polyglutamatedAntifolate and a platinum-based chemotherapeutic agent selected from:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof.In some embodiments, the Lp-αPANTIFOL comprises an alpha polyglutamatedAntifolate and an analog of a platinum-based chemotherapeutic agentselected from: stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, NDDP, anddedaplatin, or a salt or acid thereof.

In some embodiments, the liposome composition comprises liposomes thatfurther contain one or more of an immunostimulatory agent, a detectablemarker and a maleimide disposed on at least one of the PEG and theexterior of the liposome.

D Cyclodextrins

In additional embodiments, the liposome comprise a αPANTIFOL (e.g., aαPANTIFOL disclosed herein) and a cyclodextrin (e.g., a cyclodextrin inSection IIC, herein). In some embodiments, the liposome comprises aαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the liposome comprises a polyglutamated Antifolatedescribed in Section II. In some embodiments, the αPANTIFOL liposome isa liposomal composition according to any of [13]-[72] of the DetailedDescription. In some embodiments, the αPANTIFOL liposome is a targetedliposomal composition according to any of [13]-[72] of the DetailedDescription.

In some embodiments, the αPANTIFOL liposome comprises a complex formedby a cyclodextrin and a therapeutic agent. In some embodiments, thetherapeutic agent is a cytotoxic compound or a salt or acid thereof. Ina further embodiment, the therapeutic agent is a chemotherapeutic agentor a salt or acid thereof. In another embodiment, the therapeutic agentis a platinum-based drug. In another embodiment, the therapeutic agentis a taxane-based drug. In further embodiments, the therapeutic agent ofthe cyclodextrin/therapeutic agent complex is selected from:gemcitabine, a gemcitabine-based therapeutic agent, doxorubicin, anantifolate, an antifolate-based chemotherapeutic, or a salt or acid,acid or free base form thereof. In some embodiments, the αPANTIFOLliposome comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome comprises apolyglutamated Antifolate described in Section II. In some embodiments,the αPANTIFOL liposome is a liposomal composition according to any of[13]-[72] of the Detailed Description. In some embodiments, theαPANTIFOL liposome is a targeted liposomal composition according to anyof [13]-[72] of the Detailed Description. In additional embodiments, themolar ratio of cyclodextrin/therapeutic agent in the complex is in therange 1-10:1. In some embodiments, the molar ratio ofαPANTIFOL/therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, or 10:1. In some embodiments, the molar ratio ofαPANTIFOL/therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/therapeutic agent in the complex is: 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In additionalembodiments, the cyclodextrin//platinum-based agent complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In some embodiments, the αPANTIFOL liposome comprises αPANTIFOL and acyclodextrin/platinum-based chemotherapeutic agent complex. In someembodiments, the platinum-based chemotherapeutic agent is selected from:cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. Inother embodiments, the cyclodextrin/platinum-based chemotherapeuticagent complex comprises an analog of a cisplatin, carboplatin,oxaliplatin, or a salt or acid thereof. In some embodiments, theliposome comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome comprises apolyglutamated Antifolate described in Section II. In some embodiments,the αPANTIFOL liposome is a liposomal composition according to any of[13]-[72] of the Detailed Description. In some embodiments, theαPANTIFOL liposome is a targeted liposomal composition according to anyof [13]-[72] of the Detailed Description. In some embodiments, the molarratio of cyclodextrin/platinum-based agent in the complex is in therange 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/platinum-based agent in the complex is: 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In additionalembodiments, the cyclodextrin//platinum-based agent complex isencapsulated in a liposome.

In some embodiments, the platinum-based chemotherapeutic agent isselected from: cisplatin, carboplatin, and oxaliplatin, or a salt oracid thereof. In other embodiments, the cyclodextrin/platinum-basedchemotherapeutic agent complex comprises an analog of a cisplatin,carboplatin, oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/platinum-based agent in thecomplex is in the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/platinum-based agent in the complex is: 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1.

In further embodiments, the disclosure provides a complex containingcyclodextrin and cisplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or10:1. In some embodiments, the molar ratio of cyclodextrin/cisplatin (orcisplatin salt or acid) in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molar ratio ofcyclodextrin/cisplatin (or cisplatin salt or acid) in the complex is:1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. Inadditional embodiments, the cyclodextrin//cisplatin (or cisplatin saltor acid) complex is encapsulated in a liposome.

In another embodiment, the disclosure provides a complex containingcyclodextrin and carboplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or10:1. In some embodiments, the molar ratio of cyclodextrin/carboplatin(or carboplatin salt or acid) in the complex is 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/carboplatin (or carboplatin salt or acid) in thecomplex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In additional embodiments, the cyclodextrin/carboplatin (orcarboplatin salt or acid) complex is encapsulated in a liposome (e.g.,as described herein or otherwise known in the art).

In another embodiment, the disclosure provides a complex containingcyclodextrin and oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or10:1. In some embodiments, the molar ratio of cyclodextrin/oxaliplatin(or oxaliplatin salt or acid) in the complex is 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/oxaliplatin (or oxaliplatin salt or acid) in thecomplex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In additional embodiments, the cyclodextrin/oxaliplatin (oroxaliplatin salt or acid) complex is encapsulated in a liposome (e.g.,as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and a platinum-based chemotherapeutic agent selected from:nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol,cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin,tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine,traplatin, enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, anddedaplatin, or a salt or acid thereof. In other embodiments, thecyclodextrin/platinum-based chemotherapeutic agent complex comprises ananalog of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin,platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin,triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, or dedaplatin, or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/platinum-basedchemotherapeutic agent (or salt or acid or analog thereof) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 10:1. In someembodiments, the molar ratio of cyclodextrin/platinum-basedchemotherapeutic agent (or salt or acid or analog thereof) in thecomplex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50. In some embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In additional embodiments, thecyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) complex is encapsulated in a liposome (e.g., asdescribed herein or otherwise known in the art).

In some embodiments, the disclosure provides a composition comprising acyclodextrin/taxane-based chemotherapeutic agent complex. In someembodiments, the taxane-based chemotherapeutic agent is selected from:paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel(CTX), or a salt or acid thereof. In some embodiments, the molar ratioof cyclodextrin/taxane-based agent in the complex is in the range1-10:1. In some embodiments, the molar ratio ofcyclodextrin/taxane-based agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/taxane-based agent in the complex is 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molarratio of cyclodextrin/taxane-based agent in the complex is: 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In additional embodiments,the cyclodextrin/taxane-based agent complex is encapsulated in aliposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and paclitaxel (PTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of paclitaxel (PTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/paclitaxel(or paclitaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/paclitaxel (orpaclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/paclitaxel (or paclitaxel salt or acid) in the complex is1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In someembodiments, the molar ratio of cyclodextrin/paclitaxel (or paclitaxelsalt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In additional embodiments, thecyclodextrin/paclitaxel (or paclitaxel salt or acid) complex isencapsulated in a liposome.

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and docetaxel (DTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of docetaxel (DTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/docetaxel(or docetaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/docetaxel (ordocetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/docetaxel (or docetaxel salt or acid) in the complex is1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In someembodiments, the molar ratio of cyclodextrin/docetaxel (or docetaxelsalt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In additional embodiments, thecyclodextrin/docetaxel (or docetaxel salt or acid) complex isencapsulated in a liposome.

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and larotaxel (LTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of larotaxel (LTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/larotaxel(or larotaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/larotaxel (orlarotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, or 10:1. In some embodiments, the molar ratio ofcyclodextrin/larotaxel (or larotaxel salt or acid) in the complex is1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In someembodiments, the molar ratio of cyclodextrin/larotaxel (or larotaxelsalt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In additional embodiments, thecyclodextrin/larotaxel (or larotaxel salt or acid) complex isencapsulated in a liposome.

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and cabazitaxel (CTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of cabazitaxel (CTX), or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex isin the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 10:1. In some embodiments, themolar ratio of cyclodextrin/cabazitaxel (or cabazitaxel salt or acid) inthe complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50. In some embodiments, the molar ratio of acyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complexis: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. Inadditional embodiments, the cyclodextrin/cabazitaxel (or cabazitaxelsalt or acid) complex is encapsulated in a liposome.

The cyclodextrin of the cyclodextrin/therapeutic agent complex can bederivatized or underivatized. In some embodiments, the cyclodextrin isderivatized. In further embodiments, the cyclodextrin is a derivatizedbeta-cyclodextrin (e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD),and a sulfobutyl ether beta-CD (SBE)-beta-cyclodextrin)). In someembodiments, the cyclodextrin of the cyclodextrin/therapeutic agentcomplex is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxygroups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether groupsubstitutions of hydroxy groups. In further embodiments, thecyclodextrin of the cyclodextrin/therapeutic agent complex is aderivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfobutyl ether group substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the αPANTIFOL liposome composition is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R9 are each, independently, —H, a straight chain or branchedC₁-C₈-alkylene group, a 2-hydroxylpropyl-3-group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R8, and R9, is a straight-chain orbranched C₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the αPANTIFOL liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO3⁻ group;wherein at least one of R₁ and R2 is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the αPANTIFOL liposome comprises between 100 to100,000 of the cyclodextrin/therapeutic agent complexes.

In some embodiments, a cyclodextrin derivative of theαPANTIFOL/cyclodextrin complex and/or cyclodextrin/therapeutic agentcomplex is a cyclodextrin disclosed in U.S. Pat. Nos. 6,133,248,5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; andIntl. Appl. Publ. No. WO 02005/117911, the contents each of which isherein incorporated by reference in its priority.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a sulfoalkyl ethercyclodextrin. In some embodiments, the cyclodextrin derivative ofcomplex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDexPharma. Inc., Lenexa, Kans. Methods for preparing sulfobutylether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are knownin the art.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a compound of Formula III:

wherein R equals:

-   -   (a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0;    -   (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0;    -   (c) (H)_(21-X) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0; or    -   (d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0,        6.0-7.0, or 8.0-10.0.

Additional cyclodextrins and cyclodextrin/platinum-based therapeuticcomplexes that can be contained in the αPANTIFOL liposomes and usedaccording to the disclosed methods is disclosed in U.S. Appl. No.62/583,432, the contents of which is herein incorporated by reference itits entirety.

In some embodiments, the αPANTIFOL liposome comprises a complex of acyclodextrin and a platinum-based chemotherapeutic agent, or a saltthereof. In some embodiments, the platinum-based chemotherapeutic agentis cisplatin or a cisplatin analog. In some embodiments, theplatinum-based chemotherapeutic agent is carboplatin. In additionalembodiments, the liposome composition comprises a platinum-basedchemotherapeutic agent is selected from: carboplatin, cisplatin,oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin,tetraplatin, lipoplatin, lobaplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK 121, CI-973,DWA 2114R, NDDP, and dedaplatin. In some embodiments, the αPANTIFOLliposome comprises between 100 to 100,000 platinum-basedchemotherapeutic agent/CD complexes. In additional embodiments, theliposome composition comprises liposomes that have a diameter in therange of 20 nm to 500 nm, or 20 nm to 200 nm, or any range thereinbetween. In additional embodiments, the liposome composition comprisesliposomes that have a diameter in the range of 20 nm to 200 nm, or anyrange therein between. In some embodiments, liposomes in the compositioncomprise between 100 to 100,000 platinum.

(3) Targeted Liposomes

In some embodiments, the disclosure provides a liposomal alphapolyglutamated Antifolate composition wherein the liposome comprises analpha polyglutamated Antifolate and a targeting moiety attached to oneor both of a PEG and the exterior of the liposome, and wherein thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest. Such liposomes may generally be referred toherein as “targeted liposomes,” e.g., liposomes including one or moretargeting moieties or biodistribution modifiers on the surface of, orotherwise attached to, the liposomes. The targeting moiety of thetargeted liposomes can be any moiety or agent that is capable ofspecifically binding a desired target (e.g., an antigen target expressedon the surface of a target cell of interest). In one embodiment, thetargeted liposome specifically and preferentially binds to a target onthe surface of a target cell of interest that internalizes the targetedliposome into which the liposome encapsulated alpha polyglutamatedAntifolate (e.g., alpha pentaglutamated Antifolate or alphahexaglutamated Antifolate) exerts its cytotoxic effect. In furtherembodiments, the target cell is a cancer cell, a tumor cell or ametastatic cell. In some embodiments, the targeted liposome ispegylated.

The term “attach” or “attached” refers, for example, to any type ofbonding such as covalent bonding, ionic bonding (e.g., avidin-biotin)bonding by hydrophobic interactions, and bonding via functional groupssuch as maleimide, or linkers such as PEG. For example, a detectablemarker, a steric stabilizer, a liposome, a liposomal component, animmunostimulating agent may be attached to each other directly, by amaleimide functional group, or by a PEG-malemide group.

The composition and origination of the targeting moiety is non-limitingto the scope of this disclosure. In some embodiments, the targetingmoiety attached to the liposome is a polypeptide or peptidomimeticligand. Peptide and peptidomimetic targeting moieties include thosehaving naturally occurring or modified peptides, e.g., D or L peptides;alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptideshaving one or more amide, i.e., peptide, linkages replaced with one ormore urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclicpeptides. A peptidomimetic is a molecule capable of folding into adefined three-dimensional structure similar to a natural peptide. Insome embodiments, the peptide or peptidomimetic targeting moiety is 2-50amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50amino acids long

In some embodiments, the targeting moiety polypeptide is at least 40amino acid residues in length. In other embodiments, the targetingmoiety polypeptide is at least 50, 60, 75, 100, 125, 150, 175, 200, 250,or 300 amino acid residues in length.

In additional embodiments, the targeting moiety polypeptide such as anantibody or an antigen-binding antibody fragment that binds a targetantigen with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis.

In some embodiments, the targeting moiety is an antibody or an antibodyderivative. In other embodiments, the binding domain of the targetingmoiety polypeptide is not derived from the antigen binding domain of anantibody. In some embodiments, the targeting moiety is a polypeptidederived from a binding scaffold selected from a DARPin, affilin, andarmadillo repeat, D domain (see, e.g., WO 2016/164308), Z-domain(Affibody), adnectin, lipocalin, affilin, anticalin, knottin, fynomer,atrimer, kunitz domain (see, e.g., WO 2004/063337), CTLA4, or avimer(see, e.g., U.S. Publ. Nos. 2004/0175756, 2005/0053973, 2005/0048512,and 2006/0008844).

In additional embodiments, the targeting moiety is an antibody or aderivative of the antigen binding domain of an antibody that hasspecific affinity for an epitope on a cell surface antigen of interestexpressed on the surface of a target cell. In some embodiments, thetargeting moiety is a full-length antibody. In some embodiments, thetargeting moiety is an antigen binding portion of an antibody. In someembodiments, the targeting moiety is an scFv. In other embodiments, thetargeting moiety is a Fab. In some embodiments, the targeting moietycomprises a binding domain derived from the antigen binding domain of anantibody (e.g., an scFv, Fab, Fab′, F(ab′)2, an Fv fragment, adisulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CH1domains, an scFv, a minibody, a BiTE, a Tandab, a diabody ((VL-VH)₂ or(VH-VL)₂), a single domain antibody (e.g., an sdAb such as a nanobody(either VL or VH)), and a camelid VHH domain). In some embodiments, thetargeting moiety comprises one or more complementarity determiningregions (CDRs) of antibody origin. Examples of suitable antibody-basedtargeting moieties for the disclosed targeted liposomes include afull-length human antibody, a humanized antibody, a chimeric antibody,an antigen binding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody and a multimeric antibody. The antibody of theprovided targeted liposomes can have a combination of the abovecharacteristics. For example, a humanized antibody can be an antigenbinding fragment and can be pegylated and multimerized as well.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, theFv framework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability. Thehumanized antibody can be further modified by the substitution ofadditional residues either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 and5,639,641.

In further embodiments, the targeting moiety has specific affinity foran epitope on a surface antigen of a target cell of interest. In someembodiments, the target cell is a cancer cell. In some embodiments, thetarget cell is a tumor cell. In other embodiments, the target cell is animmune cell.

In some embodiments, the targeting moiety has specific affinity for anepitope expressed on a tumor cell surface antigen. The term “tumor cellsurface antigen” refers to an antigen that is common to a specifichyperproliferative disorder such as cancer. In some embodiments, thetargeting moiety has specific affinity for an epitope of a tumor cellsurface antigen that is a tumor associated antigen (TAA). A TAA is anantigen that is found on both tumor and some normal cells. A TAA may beexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or may be normally present atextremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells. Because of the dynamic nature of tumors,in some instances, tumor cells may express unique antigens at certainstages, and at others also express antigens that are also expressed onnon-tumor cells. Thus, inclusion of a certain marker as a TAA does notpreclude it being considered a tumor specific antigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a tumor cell surface antigen that is a tumor specific antigen (TSA).A TSA is an antigen that is unique to tumor cells and does not occur onother cells in the body. In some embodiments, the targeting moiety hasspecific affinity for an epitope of a tumor cell surface antigenexpressed on the surface of a cancer including but not limited toprimary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer(e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin'slymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma,uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladdercancer, kidney cancer, mesothelioma, and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancerand other cancers known in the art. In some embodiments, the targetingmoiety has specific affinity for an epitope of a cell surface antigenexpressed on the surface of a cell in the tumor microenvironment (e.g.,and antigen such as VEGFR and TIE1, or TIE2 expressed on endothelialcells and macrophage, respectively, or an antigen expressed on tumorstromal cells such as cancer-associated fibroblasts (CAFs) tumorinfiltrating T cells and other leukocytes, and myeloid cells includingmast cells, eosinophils, and tumor-associated macrophages (TAM).

In some embodiments, the targeted liposome αPANTIFOL composition (e.g.,TLp-αPANTIFOL or TPLp-αPANTIFOL) comprises a targeting moiety that hasspecific affinity for an epitope of a cancer or tumor cell surfaceantigen that is preferentially/differentially expressed on a target cellsuch as a cancer cell or tumor cell, compared to normal or non-tumorcells, that is present on a tumor cell but absent or inaccessible on anon-tumor cell. For example, in some situations, the tumor antigen is onthe surface of both normal cells and malignant cancer cells but thetumor epitope is only exposed in a cancer cell. As a further example, atumor cell surface antigen may experience a confirmation change in acancerous state that causes a cancer cell specific epitope to bepresent. A targeting moiety with specific affinity to an epitope on atargetable tumor cell surface antigen described herein or otherwiseknown in the art is useful and is encompassed by the disclosedcompositions and methods. In some embodiments, the tumor cell with thetumor cell surface antigen is a cancer cell. Examples of such tumor cellsurface antigens include, without limitation folate receptor alpha,folate receptor beta and folate receptor delta.

In further embodiments, the targeting moiety comprises a polypeptidetargeting moiety such as an antibody or an antigen-binding antibodyfragment and the targeting moiety has binding specificity for a folatereceptor. In some embodiments, the targeting moiety binds a folatereceptor with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis. In someembodiments, the folate receptor bound by the targeting moiety is one ormore folate receptors selected from: folate receptor alpha (FR-α),folate receptor beta (FR-β), and folate receptor delta (FR-δ). In afurther embodiment, the targeting moiety has specific affinity for atleast two antigens selected from folate receptor alpha, folate receptorbeta, and folate receptor delta. In another embodiment, the targetingmoiety has specific affinity for folate receptor alpha; folate receptorbeta; and folate receptor delta.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen that internalizes the targeting moietyupon binding. Numerous cell surface antigens that internalize bindingpartners such as antibodies upon binding are known in the art and areenvisioned to be binding targets for the targeting moieties expressed onthe targeted liposome αPANTIFOL compositions (e.g., TLp-αPANTIFOL orTPLp-αPANTIFOL) disclosed herein.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK.

In some embodiments, the targeting moiety has a specific affinity for acell surface antigen(s) derived, from or determined to be expressed on,a specific subject's cancer (e.g., tumor) such as a neoantigen.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from mannose-6-phosphatereceptor, transferrin receptor, and a cell adhesion molecule (CAM). Infurther embodiments, the targeting moiety has a specific affinity for anepitope of a CAM is selected from the group consist of: intercellularadhesion molecule (ICAM), platelet-endothelial cell adhesion molecule(PECAM), activated leukocyte cell adhesion molecule (ALCAM),B-lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesionmolecule (VCAM), mucosal vascular address in cell adhesion molecule(MAdCAM), CD44, LFA-2, LFA-3, and basigin.

A discussed herein, folate receptors (FRs) are distinct from reducedfolate carriers (RFCs) and exploit different pathways for bringingfolates and antifolates into cells. In some embodiments, the targetingmoiety specifically binds a folate receptor. In further embodiments, thetargeting moiety specifically binds a folate receptor selected fromfolate receptor alpha, folate receptor beta and folate receptor delta.Antibodies to folate receptor alpha can routinely be generated usingtechniques known in the art. Moreover, the sequences of numerousanti-folate receptor antibodies are in the public domain and/orcommercially available and are readily obtainable.

Murine antibodies against folate receptor are examples of antibodiesthat can be used as targeting moieties of the disclosed targetedliposome is a murine antibody against folate receptor. The sequence ofthese antibodies are known and are described, for example, in U.S. Pat.Nos. 5,646,253; 8,388,972; 8,871,206; and 9,133,275, and Intl. Appl.Nos. PCT/US2011/056966, and PCT/US2012/046672. For example, based on thesequences already in the public domain, the gene for the antibodies canbe synthesized and placed into a transient expression vector and theantibody was produced in HEK-293 transient expression system. Theantibody can be a complete antibody, a Fab, or any of the variousantibody variations discussed herein or otherwise known in the art.

In some embodiments, the targeted liposome (e.g., TL-αPANTIFOL orTPL-αPANTIFOL) contains from 1 to 1,000, or more than 1,000, targetingmoieties on its surface. In some embodiments, the targeted liposomecontains from 30 to 1,000, 30 to 500, 30 to 250 or 30-200, targetingmoieties, or any range therein between. In some embodiments, thetargeted liposome (e.g., TL-αPANTIFOL or TPL-αPANTIFOL) contains from 30to 1,000, or more than 1,000, targeting moieties on its surface. In someembodiments, the targeted liposome contains from 30 to 500, 30 to 250 or30-200, targeting moieties. In some embodiments, the targeted liposomecontains less than 220 targeting moieties, less than 200 targetingmoieties, or less than 175 targeting moieties. In some embodiments, thetargeting moiety is non-covalently bonded to the outside of the liposome(e.g., via ionic interaction or a GPI anchor). In some embodiments, thetargeted liposome comprises a αPANTIFOL according to any of [1]-[12] ofthe Detailed Description. In some embodiments, the targeted liposomecomprises a polyglutamate of an Antifolate disclosed in Section II,herein. In some embodiments, the targeted liposome is a liposomeaccording to any of [53]-[72] of the Detailed Description.

In some embodiments, the molecules on the outside of the targetedliposome (e.g., TL-αPANTIFOL or TPL-αPANTIFOL) include a lipid, atargeting moiety, a steric stabilizer (e.g., a PEG), a maleimide, and acholesterol. In some embodiments, the targeting moiety is covalentlybound via a maleimide functional group. In some embodiments, thetargeting moiety is covalently bound to a liposomal component or asteric stabilizer such as a PEG molecule. In some embodiments, all thetargeting moieties of the liposome are bound to one component of theliposome such as a PEG. In other embodiments, the targeting moieties ofthe targeted liposome are bound to different components of the liposome.For example, some targeting moieties may be bound to the lipidcomponents or cholesterol, some targeting moieties may be bound to thesteric stabilizer (e.g., PEG) and still other targeting moieties may bebound to a detectable marker or to another targeting moiety. In someembodiments, the outside of the targeted liposome (e.g., TL-αPANTIFOL orTPL-αPANTIFOL) further comprises one or more of an immunostimulatoryagent, a detectable marker and a maleimide disposed on at least one ofthe PEG and the exterior of the liposome. In some embodiments, thetargeted liposome comprises a αPANTIFOL according to any of [1]-[12] ofthe Detailed Description. In some embodiments, the targeted liposomecomprises a polyglutamate of an Antifolate disclosed in Section II,herein. In some embodiments, the targeted liposome is a liposomeaccording to any of [53]-[72] of the Detailed Description.

In some embodiments, the targeted liposome (e.g., TL-αPANTIFOL orTPL-αPANTIFOL) is anionic or neutral. In some embodiments, the targetedanionic or neutral liposome has a diameter in the range of 20 nm to 500nm or 20 nm to 200 nm, or any range therein between. In someembodiments, the targeted anionic or neutral liposome has a diameter inthe range of 20 nm to 500, or any range therein between. In someembodiments, the targeted anionic or neutral liposome has a diameter inthe range of 20 nm to 400. In some embodiments, the targeted anionic orneutral liposome has a diameter in the range of 20 nm to 300. In someembodiments, the targeted anionic or neutral liposome has a diameter inthe range of 20 nm to 200. In further embodiments, the targeted anionicor neutral liposome has a diameter in the range of 80 nm to 120 nm, orany range therein between. In some embodiments, the targeted liposomecomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the targeted liposome comprises apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the targeted liposome is a liposome according to any of[53]-[72] of the Detailed Description.

In other embodiments, the targeted liposome (e.g., TL-αPANTIFOL orTPL-αPANTIFOL) is cationic. In some embodiments, the targeted cationicliposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200nm, or any range therein between. In some embodiments, the targetedcationic liposome has a diameter in the range of 20 nm to 500, or anyrange therein between. In some embodiments, the targeted cationicliposome has a diameter in the range of 20 nm to 400. In someembodiments, the targeted cationic liposome has a diameter in the rangeof 20 nm to 300. In some embodiments, the targeted cationic liposome hasa diameter in the range of 20 nm to 200. In further embodiments, thetargeted cationic liposome has a diameter in the range of 80 nm to 120nm, or any range therein between. In some embodiments, the targetedliposome comprises a αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the targeted liposomecomprises a polyglutamate of an Antifolate disclosed in Section II,herein. In some embodiments, the targeted liposome is a liposomeaccording to any of [53]-[72] of the Detailed Description.

In some embodiments, the targeted liposomes comprise at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75%, w/w of the alpha polyglutamated Antifolate. In someembodiments, during the process of preparing the targeted liposomes, atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, of the starting material of alphapolyglutamated Antifolate is encapsulated (entrapped) in the targetedliposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the alpha tetraglutamated Antifolate,or any range therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphatetraglutamated Antifolate. In some embodiments, during the process ofpreparing the targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, ofthe starting material of alpha tetraglutamated Antifolate isencapsulated (entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the alpha pentaglutamated Antifolate,or any range therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphapentaglutamated Antifolate. In some embodiments, during the process ofpreparing the targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, ofthe starting material of alpha pentaglutamated Antifolate isencapsulated (entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the alpha hexaglutamated Antifolate,or any range therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the alphahexaglutamated Antifolate. In some embodiments, during the process ofpreparing the targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, ofthe starting material of alpha hexaglutamated Antifolate is encapsulated(entrapped) in the targeted liposomes.

Methods and techniques for covalently associating polypeptide targetingmoieties with a liposome surface molecule are known in the art and canreadily be applied to prepare the TL-αPANTIFOL or TPL-αPANTIFOL liposomecompositions.

Chemical binding of non-proteinaceous targeting moieties and othercompositions to the liposomal surface may be employed. Thus, anon-proteinaceous moiety, may be covalently or non-covalently linked to,embedded or adsorbed onto the liposome using any linking or bindingmethod and/or any suitable chemical linker known in the art. The exacttype and chemical nature of such cross-linkers and cross linking methodsis preferably adapted to the type of affinity group used and the natureof the liposome. Methods for binding or adsorbing or linking thetargeting moiety are also well known in the art. For example, in someembodiments, the targeting moiety may be attached to a group at theinterface via, but not limited to, polar groups such as amino, SH,hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides. Inaddition, the targeting moiety may be attached via, but not limited to,active groups such as succinimidyl succinate, cyanuric chloride, tosylactivated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH,Epoxy, Thiopropyl, Activated Thiol, etc., Moreover, the targeting moietymay be attached via, but not limited to, hydrophobic bonds (Van DerWaals) or electrostatic interactions that may or may not includecross-linking agents (e.g., bivalent anions, poly-anions, poly-cationsetc.).

(4) Manufacture of Liposomes

In some embodiments, the disclosure provides a method of making aliposomal composition disclosed herein. In one embodiment, the methodincludes forming a mixture comprising: (1) a liposomal component; and(2) an alpha polyglutamated (e.g., pentaglutamated or hexaglutamated)Antifolate in aqueous solution. In further embodiments, the mixturecomprises a pegylated liposomal component. The mixture is thenhomogenized to form liposomes in the aqueous solution. Further, themixture can be extruded through a membrane to form liposomes enclosingthe alpha polyglutamated Antifolate in an aqueous solution. It isunderstood the liposomal components of this disclosure can comprise anylipid (including cholesterol) including functionalized lipids and lipidsattached to targeting moieties, detectable labels, and stericstabilizers, or any subset of all of these. It is further noted that thebioactive alpha polyglutamated Antifolate in aqueous solution cancomprise any reagents and chemicals discussed herein or otherwise knownin the art for the interior or exterior of the liposome including, forexample, buffers, salts, and cryoprotectants.

In some embodiments, the disclosure provides a method of making atargeted pegylated liposomal alpha polyglutamated Antifolate(targeted-PLp-αPANTIFOL) or non-targeted PLp-αPANTIFOL disclosed herein.In one embodiment, the method includes forming a mixture comprising: (1)a liposomal component; (2) an alpha polyglutamated (e.g.,pentaglutamated or hexaglutamated) Antifolate in aqueous solution; and(3) the targeting moiety. The mixture is then homogenized to formliposomes in the aqueous solution. Further, the mixture may be extrudedthrough a membrane to form liposomes enclosing the targeted alphapolyglutamated Antifolate in an aqueous solution. It is understood thatthe targeted pegylated liposomal components can comprise any lipid(including cholesterol) including functionalized lipids and lipidsattached to targeting moieties, detectable labels, and stericstabilizers, or any subset of all of these. It is further noted that thetargeted pegylated liposome can comprise any reagents and chemicalsdiscussed herein or otherwise known in the art for the interior orexterior of the liposome including, for example, buffers, salts, andcryoprotectants.

The above methods optionally further comprise the step of lyophilizingthe composition after the removing step (discussed below) to form alyophilized composition. As stated above, targeted-PTPLA ornon-targeted-PTPLA in aqueous solution may comprise a cryoprotectantdescribed herein or otherwise known in the art. If the composition is tobe lyophilized, a cryoprotectant may be preferred.

Additionally, after the lyophilizing step, the method optionally furthercomprises the step of reconstituting the lyophilized composition bydissolving the composition in a solvent after the lyophilizing step.Methods of reconstitution are known in the art. One preferred solvent iswater. Other preferred solvents include saline solutions and bufferedsolutions.

While certain exemplary embodiments, are discussed herein, it isunderstood that liposomes can be made by any method that is known in theart. See, for example, G. Gregoriadis (editor), Liposome Technology,vol. 1-3, 1st edition, 1983; 2nd edition, 1993, CRC Press, 45 BocaRaton, Fla. Examples of methods suitable for making liposomecompositions include extrusion, reverse phase evaporation, sonication,solvent (e.g., ethanol) injection, microfluidization, detergentdialysis, ether injection, and dehydration/rehydration. The size ofliposomes can routinely be controlled by controlling the pore size ofmembranes used for low pressure extrusions or the pressure and number ofpasses utilized in microfluidization or any other suitable methods knownin the art.

In general, the alpha polyglutamated Antifolate is contained inside,that is, in the inner (interior) space of the liposomes. In oneembodiment, polyglutamated Antifolate is partially or substantiallycompletely removed from the outer medium surrounding the liposomes. Suchremoval can be accomplished by any suitable means known in the art(e.g., dilution, ion exchange chromatography, size exclusionchromatography, dialysis, ultrafiltration, and precipitation).Accordingly, the methods of making liposomal compositions set forthabove or otherwise known in the art can optionally further comprise thestep of removing alpha polyglutamated Antifolate in aqueous solutionoutside of the liposomes after forming the liposomes, for example, bythe homogenization or by the extruding step.

In other embodiments, the disclosure provides a targeted pegylatedliposomal alpha polyglutamated Antifolate (PLp-αPANTIFOL) thatselectively targets folate receptors comprising: a liposome including aninterior space, an alpha polyglutamated Antifolate disposed within theinterior space, a steric stabilizer molecule attached to an exterior ofthe liposome, and a targeting moiety comprising a protein with specificaffinity for at least one folate receptor, said targeting moietyattached to at least one of the steric stabilizer and the exterior ofthe liposome. The components of this embodiment, may be the same asdescribed for other embodiments, of this disclosure. For example, thetargeted pegylated liposomal alpha polyglutamated Antifolate and thesteric stabilizer which may be PEG, are as described in other parts ofthis disclosure.

In some embodiments, the disclosure provides a method of preparing atargeted composition comprising a pegylated liposome including anentrapped and/or encapsulated alpha polyglutamated Antifolate; atargeting moiety an amino acid chain, the amino acid chain comprising aplurality of amino acids, the targeting moiety having a specificaffinity for at least one type of folate receptor, the specific affinitybeing defined to include an equilibrium dissociation constant (Kd) in arange of 0.5×10⁻¹⁰ to 10×10⁻⁶ moles [0.05 nM to 10 μM] for at least onetype folate receptor, the targeting moiety attached to one or both of aPEG and an exterior of the liposome, the method comprising: forming amixture comprising: liposomal components and alpha polyglutamatedAntifolate in solution; homogenizing the mixture to form liposomes inthe solution; processing the mixture to form liposomes entrapping and/orencapsulating alpha polyglutamated Antifolate; and providing a targetingmoiety on a surface of the liposomes entrapping and/or encapsulating thealpha polyglutamated Antifolate, the targeting moiety having specificaffinity for at least one of folate receptor alpha (FR-α), folatereceptor beta (FR-β) and folate receptor delta (FR-δ). In someembodiments, the method comprising: forming a mixture comprising:liposomal components and alpha polyglutamated Antifolate in solution;forming liposomes entrapping and/or encapsulating alpha polyglutamatedAntifolate, for example by homogenizing or otherwise processing themixture to form liposomes; and providing a targeting moiety on a surfaceof the liposomes entrapping and/or encapsulating the alphapolyglutamated Antifolate, the targeting moiety having specific affinityfor at least one of folate receptor alpha (FR-α), folate receptor beta(FR-β) and folate receptor delta (FR-δ). In some embodiments, theprocessing includes one or more of: thin film hydration, extrusion,in-line mixing, ethanol injection technique, freezing-and-thawingtechnique, reverse-phase evaporation, dynamic high pressuremicrofluidization, microfluidic mixing, double emulsion, freeze-drieddouble emulsion, 3D printing, membrane contactor method, and stirring,and once the particles have been formed, the particles can have theirsizes further modified by one or more of extrusion and sonication. Insome embodiments, during the process of preparing the liposomes at least1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, of the starting material of alpha polyglutamatedAntifolate is encapsulated (entrapped) in the targeted liposomes. Insome embodiments, the liposomes are anionic or neutral. In someembodiments, the targeting moiety has the specific affinity for one ormore of: folate receptor alpha (FR-α), folate receptor beta (FR-β) andfolate receptor delta (FR-δ). In further embodiments, the targetingmoiety has the specific affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In additional embodiments, the targetingmoiety has the specific affinity for an epitope on a tumor cell surfaceantigen that is present on a tumor cell but absent or inaccessible on anon-tumor cell.

Liposomes can also be prepared to target particular cells, organs, orcell organelles by varying phospholipid composition or by insertingreceptors or counter-receptors into the liposomes. For example,liposomes, prepared with a high content of a nonionic surfactant, havebeen used to target the liver. (See, e.g., Japanese Patent 04-244,018 toHayakawa et al.; Kato et al., Biol. Pharm. Bull. 16:960, (1993)) Aliposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with asoybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) hasalso been shown to target the liver. (See e.g., Shimizu et al., Biol.Pharm. Bull. 20:881 (1997)).

B. Antibody Delivery Vehicles

In additional embodiments, the disclosure provides an antibody deliveryvehicle (e.g., ADC). In some embodiments, the disclosure provides animmunoconjugate having the Formula (A)-(L)-(αPANTIFOL), wherein: (A) isan antibody or antigen binding fragment of an antibody; (L) is a linker;and “(αPANTIFOL)” is a αPANTIFOL composition described herein; andwherein said linker (L) links (A) to (αPANTIFOL). In some embodiments,the polyglutamated antifolate is a αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, thepolyglutamated Antifolate is an Antifolate described in Section II,herein.

In some embodiments, the antibody or antigen binding antibody fragmenthas specific affinity for an epitope of a cell surface antigen on a cellof interest (e.g., an epitope and/or antigen described herein). Incertain embodiments, the antibody binds to an antigen target that isexpressed in or on the cell membrane (e.g., on the cell surface) of acancer/tumor and the antibody is internalized by the cell after bindingto the (antigen) target, after which the αPANTIFOL is releasedintracellularly. In some embodiments, the antibody is a full lengthantibody.

The antibody or antigen binding antibody fragment of the(A)-(L)-(αPANTIFOL) immunoconjugate can be an IgA, IgD, IgE, IgG or IgMantibody. The different classes of immunoglobulins have different andwell known subunit structures and three-dimensional configurations. Incertain embodiments, the antibody is an IgG antibody. In someembodiments, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. Incertain embodiments, the antibody is an IgG1 antibody.

In some embodiments, (A) is an antigen binding fragment of an antibody.In some embodiments, (A) is an antigen binding fragment of an antibody.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a αPANTIFOL, to an antibody or antigen bindingfragment of an antibody in a stable, covalent manner. The linkers can besusceptible to or be substantially resistant to acid-induced cleavage,light-induced cleavage, peptidase-induced cleavage, esterase-inducedcleavage, and disulfide bond cleavage, at conditions under which thecompound or the antibody remains active. Suitable linkers are known inthe art and include, for example, disulfide groups, thioether groups,acid labile groups, photolabile groups, peptidase labile groups andesterase labile groups. Linkers also include charged linkers, andhydrophilic forms thereof.

In some embodiments, the linker is selected from a cleavable linker, anon-cleavable linker, a hydrophilic linker, and a dicarboxylic acidbased linker. In another embodiment, the linker is a non-cleavablelinker. In another embodiment, the linker is selected from the groupconsisting: N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP);N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl4-(maleimidomethyl) cyclohexane-carboxylate (SMCC); N-sulfosuccinimidyl4-(maleimidomethyl) cyclohex-anecarboxylate (sulfoSMCC);N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-ma-leimide). In a further embodiment, the linker isN-succinimidyl-[(N-maleimido-propionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide).

In some embodiments, the alpha polyglutamated Antifolate is attached(coupled) to the antibody or antigen binding antibody fragment of theimmunoconjugate directly, or through a linker using techniques known inthe art. Such attachment of one or more αPANTIFOL can include manychemical mechanisms, such as covalent binding, affinity binding,intercalation, coordinate binding and complexation. Covalent binding ofthe αPANTIFOL and antibody or antigen binding antibody fragment can beachieved by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent agents are useful in associating polypeptides to otherproteins with coupling agents such as carbodiimides, diisocyanates,glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list isnot intended to be exhaustive of the various coupling agents known inthe art but, rather, is exemplary of the more common coupling agents. Insome embodiments, the antibody or antigen binding antibody fragment isderivatized and then attached to the alpha polyglutamated Antifolate.Alternatively, the αPANTIFOL can be derivatized and attached to theantibody or antigen binding antibody fragment using techniques known inthe art.

In some embodiments, the immunoconjugate comprises an antibody or anantigen-binding fragment of an antibody and a αPANTIFOL containing 4, 5,6, 2-10, 4-6, or more than 5, glutamyl groups (including the glutamylgroup of the Antifolate). In some embodiments, the immunoconjugatecomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the immunoconjugate comprises apolyglutamate of an Antifolate described in Section II, herein. In someembodiments, the immunoconjugate comprises an alpha polyglutamatedAntifolate that comprises two or more glutamyl groups in the L-form. Inother embodiments, the immunoconjugate comprises an alpha polyglutamatedAntifolate that comprises a glutamyl group in the D-form. In furtherembodiments, the immunoconjugate comprises an alpha polyglutamatedAntifolate that comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In additional embodiments, theimmunoconjugate comprises an alpha polyglutamated Antifolate thatcomprises two or more glutamyl groups that have a gamma carboxyllinkage. In some embodiments, the immunoconjugate comprises αpentaglutamated Antifolate. In further embodiments, the immunoconjugatecomprises L-α pentaglutamated Antifolate, a D-α pentaglutamatedAntifolate, or an L- and D-α pentaglutamated Antifolate. In someembodiments, the immunoconjugate comprises a α hexaglutamated Antifolate(Lp-αPANTIFOL). In further embodiments, the immunoconjugate comprises anL-α hexaglutamated Antifolate, a D-α hexaglutamated Antifolate, or an L-and D-α hexaglutamated Antifolate.

In some embodiments, the antibody delivery vehicle composition comprisesan alpha polyglutamated Antifolate and an antibody or an antigen bindingantibody fragment that has specific affinity for an epitope on a cellsurface antigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen derived from,or determined to be expressed on, a specific subject's cancer (tumor)such as a neoantigen. In some embodiments, the antibody delivery vehiclecomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Detailed Description.

In some embodiments, the antibody delivery vehicle composition comprisesan alpha polyglutamated Antifolate and an antibody or an antigen bindingantibody fragment that has specific affinity for an epitope on anantigen selected from mannose-6-phosphate receptor, transferrinreceptor, and a cell adhesion molecule (CAM). In further embodiments,the targeting moiety has a specific affinity for an epitope of a CAM isselected from the group consist of: intercellular adhesion molecule(ICAM), platelet-endothelial cell adhesion molecule (PECAM), activatedleukocyte cell adhesion molecule (ALCAM), B-lymphocyte cell adhesionmolecule (BL-CAM), vascular cell adhesion molecule (VCAM), mucosalvascular address in cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3,and basigin. In some embodiments, the antibody delivery vehiclecomposition comprises an alpha polyglutamated Antifolate according toany of [1]-[12] of the Detailed Description.

In some embodiments, the antibody delivery vehicle composition comprises1, 2, 3, 4, 5, 5-10, or greater than 10 α polyglutamated Antifolate. Insome embodiments, the antibody delivery vehicle composition comprises 1,2, 3, 4, 5, 5-10, or greater than 10, α pentaglutamated Antifolate. Insome embodiments, the antibody delivery vehicle composition comprises 1,2, 3, 4, 5, 5-10, or greater than 10, a hexaglutamated Antifolate. Insome embodiments, the antibody delivery vehicle composition comprises analpha polyglutamated Antifolate according to any of [1]-[12] of theDetailed Description.

IV. Pharmaceutical Compositions and Administration

In some embodiments, the liposome composition is provided as apharmaceutical composition containing the liposome and a carrier, e.g.,a pharmaceutically acceptable carrier. In some embodiments, the liposomecomposition is a liposome according to any of [13]-[72] of the DetailedDescription. Examples of pharmaceutically acceptable carriers containedin the provided pharmaceutical compositions include normal saline,isotonic dextrose, isotonic sucrose, Ringer's solution, and Hanks'solution. In some embodiments, a buffer substance is added to maintainan optimal pH for storage stability of the pharmaceutical composition.In some embodiments, the pH of the pharmaceutical composition is between6.0 and 7.5. In some embodiments, the pH is between 6.3 and 7.0. Infurther embodiments, the pH is 6.5. Ideally the pH of the pharmaceuticalcomposition allows for both stability of liposome membrane lipids andretention of the entrapped entities. Histidine,hydroxyethylpiperazine-ethylsulfonate (HEPES), morpholipoethylsulfonate(MES), succinate, tartrate, and citrate, typically at 2-20 mMconcentration, are exemplary buffer substances. Other suitable carriersinclude, e.g., water, buffered aqueous solution, 0.4% NaCl, and 0.3%glycine. Protein, carbohydrate, or polymeric stabilizers and tonicityadjusters can be added, e.g., gelatin, albumin, dextran, orpolyvinylpyrrolidone. The tonicity of the composition can be adjusted tothe physiological level of 0.25-0.35 mol/kg with glucose or a more inertcompound such as lactose, sucrose, mannitol, or dextrin. Thesecompositions can routinely be sterilized using conventional,sterilization techniques known in the art (e.g., by filtration). Theresulting aqueous solutions can be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous medium prior to administration.

The provided pharmaceutical liposome compositions can also contain otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, and tonicity adjusting agents, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, and calciumchloride. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alpha-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The liposome concentration in the provided fluid pharmaceuticalformulations can vary widely depending upon need, e.g., from less thanabout 0.05% usually or at least about 2-10% to as much as 30-50% byweight and will be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension. Alternatively, liposome pharmaceutical compositionscomposed of irritating lipids may be diluted to low concentrations tolessen inflammation at the site of administration.

Some embodiments, relate to a method of delivering a targeted pegylatedliposomal formulation of alpha polyglutamated Antifolate, to a tumorexpressing folate receptor on its surface. An exemplary method comprisesthe step of administering a liposome pharmaceutical composition providedherein in an amount to deliver a therapeutically effective dose of thetargeted pegylated liposomal alpha polyglutamated Antifolate to thetumor. In some embodiments, the liposomal pharmaceutical compositioncomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the liposomal composition comprises apolyglutamate of an Antifolate described in Section II, herein. In someembodiments, the liposomal pharmaceutical composition comprises aliposome composition according to any of [13]-[72] of the DetailedDescription.

The amount of liposome pharmaceutical composition administered willdepend upon the particular alpha polyglutamated Antifolate entrappedinside the liposomes, the disease state being treated, the type ofliposomes being used, and the judgment of the clinician. Generally theamount of liposome pharmaceutical composition administered will besufficient to deliver a therapeutically effective dose of the particulartherapeutic entity.

The quantity of liposome pharmaceutical composition necessary to delivera therapeutically effective dose can be determined by routine in vitroand in vivo methods, common in the art of drug testing. See, forexample, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbookof Anticancer Drug Development, L W W, 2003. Therapeutically effectivedosages for various therapeutic compositions are known to those skilledin the art. In some embodiments, a therapeutic entity delivered via thepharmaceutical liposome composition and provides at least the same orhigher activity than the activity obtained by administering the sameamount of the therapeutic entity in its routine non-liposomeformulation. Typically the dosages for the liposome pharmaceuticalcomposition is in a range for example, between about 0.005 and about5000 mg of the therapeutic entity per square meter of body surface areamost often, between about 0.1 and about 1000 mg therapeutic entity persquare meter of body surface area.

For example, if the subject has a tumor, an effective amount may be thatamount of the agent (e.g., alpha polyglutamated Antifolate composition)that reduces the tumor volume or load (as for example determined byimaging the tumor). Effective amounts can also routinely be assessed bythe presence and/or frequency of cancer cells in the blood or other bodyfluid or tissue (e.g., a biopsy). If the tumor is impacting the normalfunctioning of a tissue or organ, then the effective amount canroutinely be assessed by measuring the normal functioning of the tissueor organ. In some instances the effective amount is the amount requiredto lessen or eliminate one or more, and preferably all, symptoms.

Pharmaceutical compositions comprising the alpha polyglutamatedAntifolate compositions (e.g., liposomes containing a pentaglutamated orhexaglutamated Antifolate) are also provided. Pharmaceuticalcompositions are sterile compositions that comprise a sample liposomeand preferably alpha polyglutamated Antifolate, preferably in apharmaceutically-acceptable carrier.

Unless otherwise stated herein, a variety of administration routes areavailable. The particular mode selected will depend, upon the particularactive agent selected, the particular condition being treated and thedosage required for therapeutic efficacy. The provided methods can bepracticed using any known mode of administration that is medicallyacceptable and in accordance with good medical practice. In someembodiments, the administration route is an injection. In furtherembodiments, the injection is by a parenteral route elected from anintramuscular, subcutaneous, intravenous, intraarterial,intraperitoneal, intraarticular, intraepidural, intrathecal,intravenous, intramuscular, or intra sternal injection. In someembodiments, the administration route is an infusion. In additionalembodiments, the administration route is oral, nasal, mucosal,sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical,transdermal, pulmonary, or inhalation.

Therapeutic compositions containing αPANTIFOL compositions such as theliposomal αPANTIFOL compositions described herein can be conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionprovided herein refers to physically discrete units suitable as unitarydosage for the subject, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent; e.g., carrier, or vehicle. In aspecific embodiment, therapeutic compositions containing an Adapter areadministered subcutaneously.

In some embodiments, the α-PANTIFOL composition is administered in amanner compatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered depends on the subjectto be treated, capacity of the subject's system to utilize the activeingredient, and degree of therapeutic effect desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The αPANTIFOL composition are formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular patient being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thedosage ranges for the administration of αPANTIFOL composition are thoselarge enough to produce the desired effect in which the disease symptomsmediated by the target molecule are ameliorated. The dosage should notbe so large as to cause adverse side effects, such as, hyperviscositysyndromes, pulmonary edema, congestive heart failure, and other adverseside effects known in the art. Generally, the dosage will vary with theage, weight, height, body surface area, state of health (e.g., renal andliver function), condition, sex and extent of the disease in the patientand can routinely be determined by one of ordinary skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication.

The dosage schedule and amounts effective for therapeutic andprophylactic uses, i.e., the “dosing regimen,” will depend upon avariety of factors, including the cause, stage and severity of thedisease or disorder, the health, physical status, age of the subjectbeing treated, and the site and mode of the delivery of the αPANTIFOLcomposition. Therapeutic efficacy and toxicity of the αPANTIFOLcomposition can be determined by standard pharmaceutical,pharmacological, and toxicological procedures in cell cultures orexperimental animals. Data obtained from these procedures can likewisebe used in formulating a range of dosages for human use. Moreover,therapeutic index (i.e., the dose therapeutically effective in 50percent of the population divided by the dose lethal to 50 percent ofthe population (ED50/LD50)) can readily be determined using knownprocedures. The dosage is preferably within a range of concentrationsthat includes the ED50 with little or no toxicity, and may vary withinthis range depending on the dosage form employed, sensitivity of thepatient, and the route of administration.

The dosage regimen also takes into consideration pharmacokineticsparameters known in the art, such as, drug absorption rate,bioavailability, metabolism and clearance (see, e.g., Hidalgo-Aragones,J. Steroid Biochem. Mol. Biol. 58:611-617 (1996); Groning et al.,Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996);and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)). It is wellwithin the state of the art for the clinician to determine the dosageregimen for each subject being treated. Moreover, single or multipleadministrations of the αPANTIFOL composition can be administereddepending on the dosage and frequency as required and tolerated by thesubject. The duration of prophylactic and therapeutic treatment willvary depending on the particular disease or condition being treated.Some diseases are amenable to acute treatment whereas others requirelong-term, chronic therapy. The αPANTIFOL composition can beadministered serially, or simultaneously with the additional therapeuticagent.

In some embodiments, the αPANTIFOL composition is administered in aliposomal composition at a dose of between 0.005 and 5000 mg ofαPANTIFOL per square meter of body surface area, or any range thereinbetween. In further embodiments, the αPANTIFOL composition isadministered in a liposomal composition at a dose of between 0.1 and1000 mg αPANTIFOL/meter squared of body surface area, or any rangetherein between.

In some embodiments, the αPANTIFOL composition is administered in animmunoconjugate composition at a dose of 1 mg/kg to 500 mg/kg, 1 mg/kgto 250 mg/kg, 1 mg/kg to 200 mg/kg, 1 mg/kg to 150 mg/kg, 1 mg/kg to 100mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 25 mg/kg, 1 mg/kg to 20 mg/kg, 1mg/kg to 15 mg/kg, 1 mg/kg to 10 mg/kg, or 1 mg/kg to 5 mg/kg, or anyrange therein between.

In another embodiment, the αPANTIFOL composition is administered incombination with one or more additional therapeutics.

In some embodiment, the PLp-αPANTIFOL and/or targeted-PLp-αPANTIFOL isprepared as an infusion composition, an injection composition, aparenteral composition, or a topical composition. In furtherembodiments, the injection includes one or more of: intraperitonealinjection, direct intratumor injection, intra-arterial injection, andintravenous injection, subcutaneous injection, intramuscular injection,delivery via transcutaneous and intranasal route. In a furtherembodiment, the PLp-αPANTIFOL and/or targeted-PLp-αPANTIFOL is a liquidsolution or a suspension. However, solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection are also providedherein. In some embodiments, the targeted pegylated liposomal alphapolyglutamated Antifolate composition is formulated as an enteric-coatedtablet or gel capsule according to methods known in the art.

In some embodiments, the targeted pegylated liposomal alphapolyglutamated Antifolate formulations are administered to a tumor ofthe central nervous system using a slow, sustained intracranial infusionof the liposomes directly into the tumor (e.g., a convection-enhanceddelivery (CED)). See, Saito et al., Cancer Research 64:2572-2579 (2004);Mamot et al., J. Neuro-Oncology 68:1-9 (2004). In other embodiments, theformulations are directly applied to tissue surfaces. Sustained release,pH dependent release, and other specific chemical or environmentalcondition-mediated release administration of the pegylated liposomalalpha polyglutamated Antifolate formulations (e.g., depot injections anderodible implants) are also provided. Examples of such release-mediatingcompositions are further described herein or otherwise known in the art.

For administration by inhalation, the compositions can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoro-ethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount.

When it is desirable to deliver the compositions systemically, they canformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers. Pharmaceutical parenteral formulations include aqueoussolutions of the ingredients. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of liposomes can be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the non-targeted or targeted pegylated liposomal alphapolyglutamated Antifolate can be in powder form or lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The provided compositions (e.g., alpha polyglutamated Antifolate andliposomes containing the alpha polyglutamated Antifolate) can also beformulated in rectal or vaginal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Methods of Use and Treatment

In additional embodiments, the disclosure provides methods of usingalpha polyglutamated Antifolate (αPANTIFOL) compositions. In someembodiments, the alpha αPANTIFOL compositions are used to treat adisease or disorder.

In some embodiments, the disclosure provides a method of killing a cellthat comprises contacting the cell with a composition comprising analpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein).In some embodiments, the alpha polyglutamated Antifolate is an αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the polyglutamated Antifolate is a polyglutamate of anAntifolate described in Section II, herein. In some embodiments, thecell is contacted with a liposomal composition that contains a liposomeaccording to any of [13]-[72] of the Detailed Description. In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the hyperproliferative cell is a cancer cell. In yet furtherembodiments, the cancer cell is a primary cell or a cell from a cellline obtained/derived from a cancer selected from: a non-hematologicmalignancy including such as for example, lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In yet further embodiments, the cancer cell isa primary cell or a cell from a cell line obtained/derived from a cancerselected from breast cancer, head and neck cancer, lung cancer, stomachcancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma)choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, softtissue sarcoma (desmoid tumors, aggressive fibromatosis), bladdercancer, and central nervous system (CNS) cancer. In some embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from lung cancer (e.g., NSCLC or mesothelioma). In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from breast cancer (e.g., HER2++ or triplenegative breast cancer). In some embodiments, the contacted cancer cellis a primary cell or a cell from a cell line obtained/derived fromcolorectal cancer. In some embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from ovariancancer. In some embodiments, the contacted cancer cell is a primary cellor a cell from a cell line obtained/derived from endometrial cancer. Insome embodiments, the contacted cancer cell is a primary cell or a cellfrom a cell line obtained/derived from pancreatic cancer. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from liver cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from head and neck cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from osteosarcoma. In some embodiments, the method isperformed in vivo. In other embodiments, the method is performed invitro. In some embodiments, the αPANTIFOL composition contains 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theαPANTIFOL composition comprises an alpha pentaglutamated Antifolate. Insome embodiments, the αPANTIFOL composition comprises an alphahexaglutamated Antifolate. In some embodiments, the αPANTIFOLcomposition comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups. In some embodiments, the αPANTIFOL composition comprises D alphapolyglutamated Antifolate. In some embodiments, the αPANTIFOLcomposition comprises 2, 3, 4, 5, or more than 5, D-alpha glutamylgroups. In some embodiments, the αPANTIFOL composition comprises L and Dalpha polyglutamated Antifolate. In some embodiments, the αPANTIFOLcomposition comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containing alphapolyglutamated Antifolate (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOLdisclosed herein). In some embodiments, the cell is contacted with aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the liposome is pegylated (e.g., PLp-αPANTIFOL andNTPLp-αPANTIFOL). In some embodiments, the liposome comprises atargeting moiety on its surface that has specific affinity for anepitope of an antigen on the surface of the cell (e.g., TLp-αPANTIFOLand TPLp-αPANTIFOL). In further embodiments, the liposome is pegylatedand comprises a targeting moiety on its surface that specifically bindsan antigen on the surface of the cell (e.g., TPLp-αPANTIFOL). In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In additionalembodiments, the contacted cell is a hyperproliferative cell. In furtherembodiments, the hyperproliferative cell is a cancer cell. In furtherembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from a cancer selected from: lung cancer(e.g., non-small cell), pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma. In some embodiments, the contacted cancer cell is a primarycell or a cell from a cell line obtained/derived from lung cancer (e.g.,NSCLC or mesothelioma). In some embodiments, the contacted cancer cellis a primary cell or a cell from a cell line obtained/derived frombreast cancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from colorectal cancer. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from ovarian cancer. In some embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from endometrial cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from pancreatic cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from liver cancer. In some embodiments, the contactedcancer cell is a primary cell or a cell from a cell lineobtained/derived from head and neck cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from osteosarcoma. In some embodiments, the method isperformed in vivo. In other embodiments, the method is performed invitro. In some embodiments, the liposome contains an αPANTIFOLcontaining 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome comprises 2, 3, 4, 5, or more than 5, L-alphaglutamyl groups. In some embodiments, the liposome comprises D alphapolyglutamated Antifolate. In some embodiments, the liposome comprises2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the liposome comprises L and D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5,D-alpha glutamyl groups.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell that comprises contacting a hyperproliferativecell with a delivery vehicle (e.g., a liposome or antibody) comprisingalpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein).In some embodiments, the alpha polyglutamated Antifolate is an αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the alpha polyglutamated Antifolate is a polyglutamate ofan Antifolate described in Section II, herein. In some embodiments, thedelivery vehicle is an antibody (e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, the hyperproliferative cell is contacted with aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the delivery vehicle is non-targeted. In otherembodiments, the delivery vehicle is targeted and comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of the hyperproliferative cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on an antigen on the surface of thehyperproliferative cell selected from GONMB, TACSTD2 (TROP2), CEACAM5,EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β orfolate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety thatspecifically binds a cell surface antigen(s) derived from, or determinedto be expressed on, a specific subject's cancer (tumor) such as aneoantigen. In some embodiments, the method is performed in vivo. Insome embodiments, the method is performed in vitro. In some embodiments,the delivery vehicle comprises an αPANTIFOL consisting of 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, the deliveryvehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the delivery vehicle comprises L alphapolyglutamated Antifolate. In some embodiments, the delivery vehiclecomprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups. In someembodiments, the delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the delivery vehicle comprises 2, 3, 4,5, or more than 5, D-alpha glutamyl groups. In some embodiments, thedelivery vehicle comprises L and D alpha polyglutamated Antifolate. Insome embodiments, the delivery vehicle comprises 2, 3, 4, 5, or morethan 5, L-alpha glutamyl groups. In some embodiments, the deliveryvehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groupsand 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In particular embodiments, the method of a killing a hyperproliferativecell is performed using a liposome delivery vehicle that comprises analpha polyglutamated Antifolate (e.g., an Lp-αPANTIFOL such as,PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL orTPLp-αPANTIFOL disclosed herein). In some embodiments, thehyperproliferative cell is contacted with a liposome according to any of[13]-[72] of the Detailed Description. In some embodiments, the deliveryvehicle is a non-targeted liposome. In some embodiments, the deliveryvehicle comprises a targeting moiety on its surface that has specificaffinity for an epitope on an antigen on the surface of thehyperproliferative cell (e.g., TLp-αPANTIFOL and TPLp-αPANTIFOL). Insome embodiments, the delivery vehicle is a liposome comprising atargeting moiety on its surface that has specific affinity for anepitope on an antigen on the surface of the hyperproliferative cell. Infurther embodiments, the targeting moiety has specific affinity for anepitope on an antigen selected from GONMB, TACSTD2 (TROP2), CEACAM5,EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β orfolate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the liposome comprises a targeting moiety that specificallybinds a cell surface antigen(s) derived from, or determined to beexpressed on, a specific subject's cancer (tumor) such as a neoantigen.In some embodiments, the liposome is pegylated (e.g., PLp-αPANTIFOL, andNTPLp-αPANTIFOL). In further embodiments, the liposome is pegylated andcomprises a targeting moiety on its surface that has specific affinityfor an epitope on an antigen on the surface of the hyperproliferativecell (e.g., TPLp-αPANTIFOL). In other embodiments, the embodiments, theliposome is not pegylated. In some embodiments, the liposome is notpegylated and the liposome comprises a targeting moiety on its surfacethat has specific affinity for an epitope on an antigen on the surfaceof the hyperproliferative cell (e.g., TPLp-αPANTIFOL). In someembodiments, the liposome comprises an αPANTIFOL consisting of 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theliposome comprises an alpha tetraglutamated Antifolate. In someembodiments, the liposome comprises an alpha pentaglutamated Antifolate.In other embodiments, the liposome comprises an alpha hexaglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, the liposomecomprises D alpha polyglutamated Antifolate. In some embodiments, theliposome comprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.In some embodiments, the liposome comprises L and D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5,D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method ofinhibiting the proliferation of a cancer cell that comprises contactingthe cancer cell with a delivery vehicle (e.g., a liposome or antibody)comprising alpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosedherein). In some embodiments, the alpha polyglutamated Antifolate is anαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the alpha polyglutamated Antifolate is apolyglutamated of an Antifolate disclosed in Section II, herein. In someembodiments, the delivery vehicle is a liposome according to any of[13]-[72] of the Detailed Description. In some embodiments, the deliveryvehicle is an antibody (e.g., a full-length IgG antibody, a bispecificantibody, or a scFv). In some embodiments, the delivery vehicle is anantibody (e.g., a full-length IG antibody, a bispecific antibody, or ascFv). In some embodiments, the delivery vehicle is a liposome (e.g., anLp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the cancer cellis contacted with a liposome according to any of [13]-[72] of theDetailed Description. In some embodiments, the delivery vehicle is aliposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,the delivery vehicle is non-targeted. In some embodiments, the deliveryvehicle is targeted and comprises a targeting moiety on its surface thathas specific affinity for an epitope on an antigen on the surface of thecancer cell. In further embodiments, the delivery vehicle comprises atargeting moiety that has specific affinity for an epitope on a cellsurface antigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle is an antibody that has specificaffinity for an epitope on an antigen on the surface of the cancer cell.In some embodiments, the contacted cancer cell is a mammalian cell. Infurther embodiments, the contacted cancer cell is a human cell. Inadditional embodiments, the contacted cancer cell is a primary cell or acell from a cell line obtained/derived from a cancer selected from: lungcancer (e.g., non-small cell), pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma. In some embodiments, the contacted cancer cell is a primarycell or a cell from a cell line obtained/derived from lung cancer (e.g.,NSCLC or mesothelioma). In some embodiments, the contacted cancer cellis a primary cell or a cell from a cell line obtained/derived frombreast cancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from colorectal cancer. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from ovarian cancer. In some embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from endometrial cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from pancreatic cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from liver cancer. In some embodiments, the contactedcancer cell is a primary cell or a cell from a cell lineobtained/derived from head and neck cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from osteosarcoma. In some embodiments, the method isperformed in vivo. In some embodiments, the method is performed invitro. In some embodiments, the delivery vehicle is an antibody that hasspecific affinity for an epitope on one of the above-listed cell surfaceantigens. In other embodiments, the targeting vehicle is a liposome thatcomprises a targeting moiety that has specific affinity for an epitopeon the surface of the cancer cell. In other embodiments, the targetingvehicle is a liposome that comprises a targeting moiety that hasspecific affinity for an epitope on one of the above-listed cell surfaceantigens. In some embodiments, the delivery vehicle is a liposome thatis pegylated. In other embodiments, the delivery vehicle is a liposomethat is not pegylated. In some embodiments, the delivery vehiclecomprises a αPANTIFOL composition containing 4, 5, 6, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the delivery vehiclecomprises an alpha tetraglutamated Antifolate. In some embodiments, thedelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the delivery vehicle comprises L alphapolyglutamated Antifolate. In some embodiments, the delivery vehiclecomprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups. In someembodiments, the delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the delivery vehicle comprises 2, 3, 4,5, or more than 5, D-alpha glutamyl groups. In some embodiments, thedelivery vehicle comprises L and D alpha polyglutamated Antifolate. Insome embodiments, the delivery vehicle comprises 2, 3, 4, 5, or morethan 5, L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5, D-alphaglutamyl groups.

In further embodiments, the disclosure provides a method of inhibitingthe proliferation of a cancer cell that comprises contacting the cancercell with a liposome comprising alpha polyglutamated Antifolate (e.g.,an αPANTIFOL disclosed herein). In some embodiments, the cancer cell iscontacted with a liposome according to any of [13]-[72] of the DetailedDescription. In some embodiments, the liposome is non-targeted. In someembodiments, the liposome is targeted and comprises a targeting moietyon its surface that has specific affinity for an epitope on an antigenon the surface of the cancer cell. In further embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK. In some embodiments, the delivery vehicle comprises a targetingmoiety that specifically binds a cell surface antigen(s) derived from,or determined to be expressed on, a specific subject's cancer (tumor)such as a neoantigen. In some embodiments, the contacted cancer cell isa mammalian cell. In further embodiments, the contacted cancer cell is ahuman cell. In additional embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from a cancerselected from: lung cancer (e.g., non-small cell), pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colorectal cancer, esophagealcancer, cervical cancer, liver cancer, kidney cancer, biliary ductcancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the contactedcancer cell is a primary cell or a cell from a cell lineobtained/derived from lung cancer (e.g., NSCLC or mesothelioma). In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from breast cancer (e.g., HER2++ or triplenegative breast cancer). In some embodiments, the contacted cancer cellis a primary cell or a cell from a cell line obtained/derived fromcolorectal cancer. In some embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from ovariancancer. In some embodiments, the contacted cancer cell is a primary cellor a cell from a cell line obtained/derived from endometrial cancer. Insome embodiments, the contacted cancer cell is a primary cell or a cellfrom a cell line obtained/derived from pancreatic cancer. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from liver cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from head and neck cancer. In some embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from osteosarcoma. In some embodiments, the method isperformed in vivo. In some embodiments, the method is performed invitro. In other embodiments, the targeting vehicle is a liposome thatcomprises a targeting moiety that has specific affinity for an epitopeon one of the above-listed cell surface antigens. In some embodiments,the liposome is pegylated. In other embodiments, the liposome that isnot pegylated. In some embodiments, the liposome comprises an αPANTIFOLcomposition containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the liposome comprises an alphatetraglutamated Antifolate. In some embodiments, the liposome comprisesan alpha pentaglutamated Antifolate. In other embodiments, the liposomecomprises an alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups.In some embodiments, the liposome comprises D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, the liposomecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the liposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a delivery vehicle (e.g., antibody or liposome) comprisingalpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein) toa subject having or at risk of having a hyperproliferative disorder. Insome embodiments, the delivery vehicle comprises a αPANTIFOL accordingto any of [1]-[12] of the Detailed Description. In some embodiments, thedelivery vehicle comprises an alpha polyglutamated Antifolate that is apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-αPANTIFOL such as,PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, orTPLp-αPANTIFOL). In some embodiments, the delivery vehicle is a liposomeaccording to any of [13]-[72] of the Detailed Description. In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of the hyperproliferative cell. In additionalembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen selectedfrom: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g.,folate receptor-α, folate receptor-β or folate receptor-δ), Mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C(GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, PCadherin, Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin,Collagen IV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR,EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3,FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6,CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30,CD33, CD34, CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79,CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphAreceptor, an EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7,EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g.,integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA,TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1,PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2,PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, thedelivery vehicle comprises a targeting moiety that specifically binds(i.e., has specific affinity for) an epitope on a cell surface antigen acell surface antigen(s) derived from, or determined to be expressed on,a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety is an antibody or an antigen bindingantibody fragment. In some embodiments, the administered deliveryvehicle does not comprise a targeting moiety that has a specificaffinity for an epitope on a cell surface antigen of thehyperproliferative cell. In some embodiments, the administered deliveryvehicle comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises L and D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is an autoimmune disease(e.g., rheumatoid arthritis). In some embodiments, thehyperproliferative disorder is a benign or malignant tumor; leukemia,hematological, or lymphoid malignancy. In other embodiments, thehyperproliferative disorder selected from a neuronal, glial, astrocytal,hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic,inflammatory, angiogenic and immunologic disorder, including anautoimmune disease.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a liposome comprising alpha polyglutamated Antifolate (e.g.,an Lp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL) to a subject having or at risk ofhaving a hyperproliferative disorder. In some embodiments, the liposomecomprises an αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the liposome comprises an alphapolyglutamated Antifolate that is a polyglutamate of an Antifolatedisclosed in Section II, herein. In some embodiments, the liposome is aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the liposome is pegylated. In some embodiments, theliposome is not pegylated. In additional embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeof antigen on the surface of the hyperproliferative cell. In additionalembodiments, the liposome comprises a targeting moiety that has specificaffinity for an epitope on a cell surface antigen selected from: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b,CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphAreceptor, an EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7,EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g.,integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA,TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1,PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2,PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, theliposome comprises a targeting moiety that has specific affinity for anepitope on a cell surface antigen(s) derived from, or determined to beexpressed on, a specific subject's cancer (tumor) such as a neoantigen.In some embodiments, the targeting moiety is an antibody or an antigenbinding antibody fragment. In some embodiments, the liposome does notcomprise a targeting moiety that has a specific affinity for an epitopeon a cell surface antigen of the hyperproliferative cell. In someembodiments, the liposome comprises αPANTIFOL containing 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, the liposomecomprises an alpha tetraglutamated Antifolate. In some embodiments, theliposome comprises an alpha pentaglutamated Antifolate. In otherembodiments, the liposome comprises an alpha hexaglutamated Antifolate.In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5,L-alpha glutamyl groups. In some embodiments, the liposome comprises Dalpha polyglutamated Antifolate. In some embodiments, the liposomecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the liposome comprises L and D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5,D-alpha glutamyl groups. In some embodiments, the hyperproliferativedisorder is cancer. In some embodiments, the hyperproliferative disorderis an autoimmune disease (e.g., rheumatoid arthritis). In someembodiments, the hyperproliferative disorder is a benign or malignanttumor; leukemia, hematological, or lymphoid malignancy. In otherembodiments, the hyperproliferative disorder is selected from aneuronal, glial, astrocytal, hypothalamic, glandular, macrophagal,epithelial, stromal, blastocoelic, inflammatory, angiogenic andimmunologic disorder, including an autoimmune disease.

Exemplary hyperproliferative disorders that can be treated according tothe disclosed methods include, but are not limited to, disordersassociated with benign, pre-malignant, and malignant cellularproliferation, including but not limited to, neoplasms and tumors (e.g.,histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer,small cell lung cancer, gastrointestinal cancer, bowel cancer,colorectal cancer, breast carcinoma, ovarian carcinoma, prostate cancer,testicular cancer, liver cancer, kidney cancer, bladder cancer,pancreatic cancer, brain cancer, sarcoma (e.g., osteosarcoma, Kaposi'ssarcoma), and melanoma), leukemias, psoriasis, bone diseases,fibroproliferative disorders (e.g., of connective tissues), andatherosclerosis.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., antibody or liposome) comprising alpha polyglutamatedAntifolate (e.g., an αPANTIFOL disclosed herein) to a subject having orat risk of having cancer. In some embodiments, the delivery vehiclecomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the delivery vehicle comprises analpha polyglutamated Antifolate that is a polyglutamate of an Antifolatedisclosed in Section II, herein. In some embodiments, the deliveryvehicle is an antibody (e.g., a full-length IgG antibody, a bispecificantibody, or a scFv). In some embodiments, the delivery vehicle is aliposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,the delivery vehicle is a liposome according to any of [13]-[72] of theDetailed Description. In some embodiments, the administered deliveryvehicle is pegylated. In some embodiments, the administered deliveryvehicle is not pegylated. In additional embodiments, the administereddelivery vehicle comprises a targeting moiety that has a specificaffinity for an epitope of antigen on the surface of a cancer cell. Insome embodiments, the delivery vehicle comprises a targeting moiety thathas specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the targetingmoiety is an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle comprises αPANTIFOLcontaining 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises an alphatetraglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises L and D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the cancer is selected from: lung (e.g., non-small lungcancer), pancreatic, breast cancer, ovarian, lung, prostate, head andneck, gastric, gastrointestinal, colon, esophageal, cervical, kidney,biliary duct, gallbladder, and a hematologic malignancy (e.g., aleukemia or lymphoma). In some embodiments, the cancer is lung cancer(e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breastcancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the cancer is colorectal cancer. In some embodiments, thecancer is ovarian cancer. In some embodiments, the cancer is endometrialcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

In additional embodiments, the disclosure provides a method fortreating, reducing, or inhibiting metastasis that comprisesadministering an effective amount of a delivery vehicle (e.g., antibodyor liposome) comprising alpha polyglutamated Antifolate (e.g., aαPANTIFOL disclosed herein) to a subject having or at risk of havingcancer. In some embodiments, the disclosed methods provide among otherthings, (1) reducing or inhibiting growth, proliferation, survival,mobility or invasiveness of a primary tumor, cancer or neoplasia; (2)reducing or inhibiting growth, proliferation, survival, mobility orinvasiveness of a primary tumor, cancer or neoplasia that potentially ordoes develop metastases; (3) reducing or inhibiting formation orestablishment of metastases arising from a primary tumor, cancer orneoplasia to one or more other sites, locations, regions or systemsdistinct from the primary tumor, cancer or neoplasia; (4) reducing orinhibiting growth or proliferation of a metastasis at one or more othersites, locations, regions or systems distinct from the primary tumor,cancer or neoplasia after a metastasis has formed or has beenestablished; and/or (5) reducing or inhibiting formation orestablishment of additional metastasis after the metastasis has beenformed or established. In some embodiments, the delivery vehicle is anantibody (e.g., a full-length IgG antibody, a bispecific antibody, or ascFv). In some embodiments, the delivery vehicle is a liposome (e.g., anLp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the administereddelivery vehicle is pegylated. In some embodiments, the administereddelivery vehicle is not pegylated. In additional embodiments, theadministered delivery vehicle comprises a targeting moiety that has aspecific affinity for an epitope of antigen on the surface of a cancercell. In some embodiments, the targeting moiety is an antibody or anantigen binding antibody fragment. In some embodiments, the administereddelivery vehicle comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the administereddelivery vehicle comprises alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises L and D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the cancer is selected from: lung (e.g., non-small lungcancer), pancreatic, breast cancer, ovarian, lung, prostate, head andneck, gastric, gastrointestinal, colon, esophageal, cervical, kidney,biliary duct, gallbladder, and a hematologic malignancy (e.g., aleukemia or lymphoma). In some embodiments, the cancer is lung cancer(e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breastcancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the cancer is colorectal cancer. In some embodiments, thecancer is ovarian cancer. In some embodiments, the cancer is endometrialcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., antibody or liposome) comprising alpha polyglutamatedAntifolate (e.g., an αPANTIFOL disclosed herein) to a subject having orat risk of having cancer. In some embodiments, the delivery vehiclecomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the delivery vehicle comprises analpha polyglutamated Antifolate that is a polyglutamate of an Antifolatedisclosed in Section II, herein. In some embodiments, the deliveryvehicle is an antibody (e.g., a full-length IgG antibody, a bispecificantibody, or a scFv). In some embodiments, the delivery vehicle is aliposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,the delivery vehicle is a liposome according to any of [13]-[72] of theDetailed Description. In some embodiments, the administered deliveryvehicle is pegylated. In some embodiments, the administered deliveryvehicle is not pegylated. In additional embodiments, the administereddelivery vehicle comprises a targeting moiety that has a specificaffinity for an epitope of antigen on the surface of a cancer cell. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety that has specific affinity for an epitope on a cell surfaceantigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle comprises αPANTIFOLcontaining 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises an alphatetraglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises L and D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the cancer is selected from: lung (e.g., non-small lungcancer), pancreatic, breast cancer, ovarian, lung, prostate, head andneck, gastric, gastrointestinal, colon, esophageal, cervical, kidney,biliary duct, gallbladder, and a hematologic malignancy (e.g., aleukemia or lymphoma). In some embodiments, the cancer is lung cancer(e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breastcancer (e.g., HER2++ or triple negative breast cancer). In someembodiments, the cancer is colorectal cancer. In some embodiments, thecancer is ovarian cancer. In some embodiments, the cancer is endometrialcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising alpha polyglutamated Antifolate (e.g., an Lp-αPANTIFOL suchas, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, orTPLp-αPANTIFOL) to a subject having or at risk of having cancer. In someembodiments, the liposome comprises an αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomecomprises an alpha polyglutamated Antifolate that is a polyglutamate ofan Antifolate disclosed in Section II, herein. In some embodiments, theliposome is a Lp-αPANTIFOL according to any of [53]-[72] of the DetailedDescription. In some embodiments, the liposome is a liposome accordingto any of [13]-[72] of the Detailed Description. In some embodiments,the liposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK. In some embodiments, the liposome comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the cancer isselected from: lung (e.g., non-small lung cancer), pancreatic, breastcancer, ovarian, lung, prostate, head and neck, gastric,gastrointestinal, colon, esophageal, cervical, kidney, biliary duct,gallbladder, and a hematologic malignancy (e.g., a leukemia orlymphoma). In some embodiments, the cancer is lung cancer (e.g., NSCLCor mesothelioma). In some embodiments, the cancer is breast cancer(e.g., HER2++ or triple negative breast cancer). In some embodiments,the cancer is colorectal cancer. In some embodiments, the cancer isovarian cancer. In some embodiments, the cancer is endometrial cancer.In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma. In some embodiments, the targeting moiety is an antibodyor an antigen binding antibody fragment. In some embodiments, theliposome comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups. In some embodiments, the liposome comprises an alphatetraglutamated Antifolate. In some embodiments, the liposome comprisesan alpha pentaglutamated Antifolate. In other embodiments, the liposomecomprises an alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups.In some embodiments, the liposome comprises D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, the liposomecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the liposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising alpha polyglutamated Antifolate (e.g., an Lp-αPANTIFOL suchas, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, orTPLp-αPANTIFOL) to a subject having or at risk of having cancer. In someembodiments, the liposome comprises an αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomecomprises an alpha polyglutamated Antifolate that is a polyglutamate ofan Antifolate disclosed in Section II, herein. In some embodiments, theliposome is a Lp-αPANTIFOL according to any of [53]-[72] of the DetailedDescription. In some embodiments, the liposome is a liposome accordingto any of [13]-[72] of the Detailed Description. In some embodiments,the liposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK. In some embodiments, the liposome comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the cancer isselected from: lung (e.g., non-small lung cancer), pancreatic, breastcancer, ovarian, lung, prostate, head and neck, gastric,gastrointestinal, colon, esophageal, cervical, kidney, biliary duct,gallbladder, and a hematologic malignancy (e.g., a leukemia orlymphoma). In some embodiments, the cancer is lung cancer (e.g., NSCLCor mesothelioma). In some embodiments, the cancer is breast cancer(e.g., HER2++ or triple negative breast cancer). In some embodiments,the cancer is colorectal cancer. In some embodiments, the cancer isovarian cancer. In some embodiments, the cancer is endometrial cancer.In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris head and neck cancer. In some embodiments, the cancer isosteosarcoma.

In some embodiments, the targeting moiety is an antibody or an antigenbinding antibody fragment. In some embodiments, the liposome comprisesαPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the liposome comprises an alphatetraglutamated Antifolate. In some embodiments, the liposome comprisesan alpha pentaglutamated Antifolate. In other embodiments, the liposomecomprises an alpha hexaglutamated Antifolate. In some embodiments, theliposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups.In some embodiments, the liposome comprises D alpha polyglutamatedAntifolate. In some embodiments, the liposome comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, the liposomecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the liposome comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition containinga liposome that comprises an alpha polyglutamated Antifolate and atargeting moiety that has a specific affinity for an epitope of antigenon the surface of the cancer. In some embodiments, the liposomecomprises an αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, liposomes of the administeredliposomal composition comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6,or more than 5, glutamyl groups. In some embodiments, the liposomecomprises an alpha polyglutamated Antifolate that is a polyglutamate ofan Antifolate disclosed in Section II, herein. In some embodiments,liposomes of the administered liposomal composition comprise alphatetraglutamated Antifolate. In some embodiments, liposomes of theadministered liposomal composition comprise alpha pentaglutamatedAntifolate. In other embodiments, liposomes of the administeredliposomal composition comprises an alpha hexaglutamated Antifolate. Insome embodiments, the liposome is a Lp-αPANTIFOL according to any of[53]-[72] of the Detailed Description. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen selected from the group consisting: of GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b,CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphAreceptor, an EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7,EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g.,integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA,TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1,PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2,PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, theliposomal composition is administered to treat a cancer selected from:lung cancer, pancreatic, breast cancer, ovarian cancer, lung cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer,biliary duct cancer, gallbladder cancer, and a hematologic malignancy.In some embodiments, the liposomal composition is administered to treatlung cancer (e.g., NSCLC or mesothelioma). In some embodiments, theliposomal composition is administered to treat breast cancer (e.g.,HER2++ or triple negative breast cancer). In some embodiments, theliposomal composition is administered to treat colorectal cancer. Insome embodiments, the liposomal composition is administered to treatovarian cancer. In some embodiments, the liposomal composition isadministered to treat endometrial cancer. In some embodiments, theliposomal composition is administered to treat pancreatic cancer. Insome embodiments, the liposomal composition is administered to treatliver cancer. In some embodiments, the liposomal composition isadministered to treat head and neck cancer. In some embodiments, theliposomal composition is administered to treat osteosarcoma.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g.,TPLp-αPANTIFOL). In some embodiments, the administered liposomalcomposition comprises liposomes that are not pegylated.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a tumor specific antigen (TSA) ortumor associated antigen (TAA). In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof an antigen selected from: a tumor differentiation antigen (e.g.,MART1/MelanA, gp100 (Pmel 17), tyrosinase, TRP1, and TRP2), atumor-specific multilineage antigen (e.g., MAGE1, MAGE3, BAGE, GAGE1,GAGE2, and p15), an overexpressed embryonic antigen (e.g.,carcinoembryonic antigen (CEA)), an overexpressed oncogene or mutatedtumor-suppressor gene product (e.g., p53, Ras, and HER2/neu), a uniquetumor antigen resulting from chromosomal translocations (e.g., BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR), a viral antigen (e.g., EpsteinBarr virus antigen EBVA, human papillomavirus (HPV) antigen E6 or E7),GP 100), prostatic acid phosphatase (PAP), prostate-specific antigen(PSA), PTGER4, ITGA4, CD37, CD52, CD62L (L-selectin), CXCR4, CD69, EVI2B(CD361), SLC39A8, MICB, LRRC70, CLELC2B, HMHA1, LST1, and CMTM6(CKLFSF6). In some embodiments, the liposome comprises an αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the liposome is a Lp-αPANTIFOL according to any of[53]-[72] of the Detailed Description.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a hematologic tumor antigen. Infurther embodiments, the targeting moiety has specific affinity for anepitope of a hematologic tumor antigen selected from: CD19, CD20, CD22,CD30, CD138, CD33 CD34, CD38, CD123, CS1, ROR1, Lewis^(Y), Ig kappalight chain, TCR, BCMA, TACI, BAFFR (CD268), CALLA, and a NKG2DLligand). In some embodiments, the liposome comprises a targeting moietythat has specific affinity for an epitope of a B-cell lymphoma-specificidiotype immunoglobulin, or a B-cell differentiation antigen (e.g.,CD19, CD20, and CD37). In some embodiments, the liposome comprises atargeting moiety that has specific affinity for an epitope of an antigenon a multiple myeloma cell (e.g., CS-1, CD38, CD138, MUC1, HM1.24,CYP1B1, SP17, PRAME, Wilms' tumor 1 (WT1), and heat shock protein gp96)or an antigen on myeloid cells (e.g., TSLPR and IL-7R). In someembodiments, the liposome comprises an αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomeis a Lp-αPANTIFOL according to any of [53]-[72] of the DetailedDescription.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a solid tumor antigen. In furtherembodiments, the targeting moiety has specific affinity for an epitopeof a hematologic tumor antigen selected from: disialoganglioside (GD2),o-acetyl GD2, EGFRvIII, ErbB2, VEGFR2, FAP, mesothelin, IL13Ra2(glioma), cMET, PSMA, L1CAM, CEA, and EGFR. In some embodiments, theliposome comprises a targeting moiety that has specific affinity for anepitope of an antigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5,EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β orfolate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the liposome comprises a targeting moiety that has specificaffinity for an epitope of an antigen selected from: CD137, PDL1, CTLA4,CD47, KIR, TNFRSF10B (DR5), TIM3, PD1, cMet, Glycolipid F77, EGFRvIII,HLAA2 (NY-ESO-1), LAG3, CD134 (OX40), HVEM, BTLA, TNFRSF25 (DR3), CD133,MAGE A3, PSCA, MUC1, CD44v6, CD44v6/7, CD44v7/8, IL11Ra, ephA2, CAIX,MNCAIX, CSPG4, MUC16, EPCAM (EGP2), TAG72, EGP40, ErbB receptor family,ErbB2 (HER2), ErbB3/4, RAGE1, GD3, FAR, Lewis^(Y), NCAM, HLAA1/MAGE1,MAGEA1, MAGEA3, MAGE-A4, B7H3, WT1, MelanA (MART1), HPVE6, HPVE7,thyroglobulin, tyrosinase, PSA, CLL1GD3, Tn Ag, FLT3, KIT, PRSS21, CD24,PDGFR-beta, SSEA4, prostase, PAP, ELF2M, ephB2, IGF1, IGFII, IGFIreceptor, LMP2, gp100, bcr-abl, Fucosyl GM1, sLe, GM3, TGS5, folatereceptor beta, TEM1 (CD248), TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97,CD7a, HLE, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2,HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, LAGE1a, legumain, E7,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT1, MAD-CT2, Fos-relatedantigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA1(Galectin 8), Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP4, SSX2, reverse transcriptase, RU1, RU2, intestinal carboxylesterase, neutrophil elastase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRLS, IGLL1,TSP-180, MAGE4, MAGE5, MAGE6, VEGFR1, IGF1R, hepatocyte growth factorreceptor, p185ErbB2, p180ErbB-3, nm-23H1, CA 19-9, CA 72-4, CAM 17.1,NuMa, K-ras, beta-Catenin, CDK4, Mum1, p15, p16, 43-9F, 5T4, 791Tgp72,β-human chorionic gonadotropin, BCA225, BTAA, CA125, CA15-3, CA 27.29(BCAA), CA195, CA242, CA-50, CAM43, CD68, CO-029, FGF5, G250, HTgp-175,M344, MA50, MG7-Ag, MOV18, NB/70K, NY-CO1, RCAS1, SDCCAG16, M2BP, TAAL6,TLP, and TPS, glioma-associated antigen, alpha-fetoprotein (AFP), p26fragment of AFP, lectin-reactive AFP, and TLR4. In some embodiments, theliposome comprises an αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome is aLp-αPANTIFOL according to any of [53]-[72] of the Detailed Description.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from: PDGFRA,VEGFR1, VEGFR3, neuropilin 1 (NRP1), neuropilin 2 (NRP2), betacellulin,PLGF, RET (rearranged during transfection), TIE1, TIE2 (TEK), CA125,CD3, CD4, CD7, CD10, CD13, CD25 CD32, CD32b, CD44 (e.g., CD44v6), CD47,CD49e (integrin alpha 5), CD54 (ICAM), CD55, CD64, CD74, CD80, CD90,CD200, CD147, CD166, CD200, ESA, SHH, DHH, IHH, patched 1 (PTCH1),smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4, WNT4A, WNT5A, WNT5B, WNT7B,WNT8A, WNT10A, WNT10B, WNT16B, LKP5, LRP5, LRP6, FZD1, FZD2, FZD4, FZD5,FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4, DLL4, Jagged, Jagged1,Jagged2, Jagged3, TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF6(Fas, CD95), TNFRSF6B (DcR3), TNFRSF7 (CD27), TNFSF9 (41BB Ligand),TNFRSF8 (CD30), TNFRSF10A (TRAILR1, DR4), TNFRSF11A (RANK), TNFRSF12(TWEAKR), TNFRSF19L (KELT), TNFRSF19 (TROY), TNFRSF21 (DR6), ILIRI,1L1R2, IL2R, IL5R, IL6R, 1L8R, IL10R, IL12R, IL13R, IL15R, IL18R, IL19R,IL21R, IL23R, XAG1, XAG3, REGIV, FGFR1, FGFR2, FGFR3, ALK, ALK1, ALK7,ALCAM, Ax1, TGFb, TGFb2, TGFb3, TGFBR1, IGFIIR, BMPRI, N-cadherin,E-cadherin, VE-cadherin, ganglioside GM2, ganglioside GD3, PSGR, DCC,CDCP1, CXCR2, CXCR7, CCR3, CCR4, CCR5, CCR7, CCR10, Claudin1, Claudin2,Claudin3, Claudin4, TMEFF2, neuregulin, MCSF, CSF, CSFR (fins), GCSF,GCSFR, BCAM, BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5, HM 1.24, LFA1, LYNX,S100A8, S100A9, SCF, Von Willebrand factor, Lewis Y6 receptor, CA G250(CA9), CRYPTO, VLA5, HLADR, MUC18, mucin CanAg, EGFL7, integrin avb3,integrin α5β activin B1 alpha, leukotriene B4 receptor (LTB4R),neurotensin NT receptor (NTR), 5T4 oncofetal antigen, Tenascin C, MMP,MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G, SULF1, SULF2, MET,CA9, TM4SF1, syndecan (SDCl), Ephrin B4, TEM1, TGFbeta 1, and TGFBRII.In some embodiments, the liposome comprises an αPANTIFOL according toany of [1]-[12] of the Detailed Description. In some embodiments, theliposome is a Lp-αPANTIFOL according to any of [53]-[72] of the DetailedDescription.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen associated with adisorder of the immune system (e.g., an autoimmune disorder and aninflammatory disorder), or is associated with regulating an immuneresponse. In some embodiments, the targeting moiety has specificaffinity for an epitope of a cell surface antigen expressed on thesurface of a macrophage (expressing CD44). In some embodiments, theliposome comprises an αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome is aLp-αPANTIFOL according to any of [53]-[72] of the Detailed Description.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an immunoinhibitory target. Inanother embodiment, the AD is an epitope of an immunoinhibitory targetselected from: IL1Ra, IL6R, CD26L, CD28, CD80, FcGamma RIIB. In anotherembodiment, the AD in the Adapter is an epitope of an immunostimulatorytarget selected from: CD25, CD28, CTLA4, PD1, B7H1 (PDL1), B7H4 TGFbeta,TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF9 (41BB, CD137), TNFRSF14 (HVEM),TNFRSF25 (DR3), and TNFRSF18 (GITR). In some embodiments, the liposomecomprises a αPANTIFOL according to any of [1]-[12] of the DetailedDescription. In some embodiments, the liposome is a Lp-αPANTIFOLaccording to any of [53]-[72] of the Detailed Description.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from: IL1Rb,C3AR, C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8, CCR9, CCR10, ChemR23,MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TREM1, TREM2,CD49a (integrin alpha 1), integrin a5b3, alpha4 integrin subunit, A4B7integrin, cathepsin G, TNFRSF3 (LTBR), TNFRSF6 (Fas, CD95), TNFRSF6B(DcR3), TNFRSF8 (CD30), TNFRSF11A (RANK), TNFRSF16 (NGFR), TNFRSF19L(RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), CD14, CD23, CD36, CD36L, CD39,CD91, CD153, CD164, CD200, CD200R, B71 (CD80), B72 (CD86), B7h, B7DC(PDL2), ICOS, ICOSL, MHC, CD, B7H2, B7H3, B7x, SLAM, KIM1, SLAMF2,SLAMF3, SLAMF4, SLAMF5, SLAMF6, SLAMF7, TNFRSF1A (TNFR1, p55, p60),TNFRSF1B (TNFR2), TNFRSF7 (CD27), TNFRSF12 (TWEAKR), TNFRSF5 (CD40),IL1R, IL2R, IL4Ra, IL5R, IL6RIL15R, IL17R, IL17Rb, IL17RC, IL22RA,1L23R, TSLPR, B7RP1, cKit, GMCSF, GMCSFR, CD2, CD4, CD11a, CD18, CD30,CD40, CD86, CXCR3, CCR2, CCR4, CCR5, CCR8, RhD, IgE, and Rh. In someembodiments, the liposome comprises an αPANTIFOL according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomeis a Lp-αPANTIFOL according to any of [53]-[72] of the DetailedDescription.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) alpha polyglutamatedAntifolate (αPANTIFOL) and (b) a targeting moiety that has specificbinding affinity for a folate receptor. In some embodiments, theliposome comprises an αPANTIFOL according to any of [1]-[12] of theDetailed Description. In some embodiments, the liposome comprises analpha polyglutamated Antifolate that is a polyglutamate of an Antifolatedisclosed in Section II, herein. In some embodiments, the targetingmoiety has specific binding affinity for folate receptor alpha (FR-α),folate receptor beta (FR-β), and/or folate receptor delta (FR-δ). Insome embodiments, the targeting moiety has a specific binding affinityfor folate receptor alpha (FR-α), folate receptor beta (FR-β), and/orfolate receptor delta (FR-δ). In some embodiments, the targeting moietyhas a specific binding affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g.,TPLp-αPANTIFOL). In some embodiments, the administered liposomalcomposition comprises liposomes that are not pegylated. In someembodiments, liposomes of the administered liposomal compositioncomprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, liposomes of the administeredliposomal composition comprise alpha tetraglutamated Antifolate. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha pentaglutamated Antifolate. In other embodiments,liposomes of the administered liposomal composition comprises an alphahexaglutamated Antifolate. In some embodiments, the liposomalcomposition is administered to treat an epithelial tissue malignancy. Insome embodiments, the liposomal composition is administered to treat acancer selected from: lung cancer, pancreatic, breast cancer, ovariancancer, lung cancer, prostate cancer, head and neck cancer, gastriccancer, gastrointestinal cancer, colon cancer, esophageal cancer,cervical cancer, kidney cancer, biliary duct cancer, gallbladder cancer,and a hematologic malignancy. In some embodiments, the liposomalcomposition is administered to treat a cancer selected from: breastcancer, advanced head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis), bladder cancer, and centralnervous system (CNS) lymphoma. In some embodiments, the liposomalcomposition is administered to treat lung cancer (e.g., NSCLC ormesothelioma). In some embodiments, the liposomal composition isadministered to treat breast cancer (e.g., HER2++ or triple negativebreast cancer). In some embodiments, the liposomal composition isadministered to treat colorectal cancer. In some embodiments, theliposomal composition is administered to treat ovarian cancer. In someembodiments, the liposomal composition is administered to treatendometrial cancer. In some embodiments, the liposomal composition isadministered to treat pancreatic cancer. In some embodiments, theliposomal composition is administered to treat liver cancer. In someembodiments, the liposomal composition is administered to treat head andneck cancer. In some embodiments, the liposomal composition isadministered to treat osteosarcoma.

In some embodiments, the disclosure provides a method for treating lungcancer (e.g., non-small lung cancer) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising alpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosedherein) to a subject having or at risk of having lung cancer. Inparticular embodiments, the, the cancer is non-small cell lung cancer.In some embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-αPANTIFOLsuch as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL,or TPLp-αPANTIFOL). In some embodiments, the administered deliveryvehicle is pegylated. In some embodiments, the administered deliveryvehicle is not pegylated. In additional embodiments, the deliveryvehicle comprises a targeting moiety on its surface that has specificaffinity for an epitope on an antigen on the surface of a lung cancer(e.g., non-small cell lung cancer) cell. In further embodiments, thedelivery vehicle comprises a targeting moiety that has specific affinityfor an epitope on an antigen selected from Mucin 1, Nectin 4, NaPi2b,CD56, EGFR, and SC-16. In some embodiments, the targeting moiety is anantibody or a fragment of an antibody. In additional embodiments, thedelivery vehicle is a liposome, and the liposome comprises a targetingmoiety that has specific affinity for an epitope on an antigen selectedfrom Mucin 1, Nectin 4, NaPi2b, CD56, EGFR, and SC-16. In furtherembodiments, the delivery vehicle is a pegylated liposome that comprisesa targeting moiety that has specific affinity for an epitope on anantigen selected from: Mucin 1, Nectin 4, NaPi2b, CD56, EGFR, and SC-16.In some embodiments, the administered delivery vehicle comprisesαPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the administered delivery vehicle comprisesan alpha tetraglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises an alpha pentaglutamatedAntifolate. In other embodiments, the administered delivery vehiclecomprises an alpha hexaglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises L alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, D-alpha glutamylgroups. In some embodiments, the administered delivery vehicle comprisesL and D alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises 2, 3, 4, 5, or more than 5,L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5, D-alpha glutamylgroups.

In some embodiments, the disclosure provides a method for treatingpancreatic cancer that comprises administering an effective amount of adelivery vehicle (e.g., an antibody (ADC) or liposome) comprising alphapolyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein) to asubject having or at risk of having pancreatic cancer. In someembodiments, the delivery vehicle comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the delivery vehicle comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, thedelivery vehicle is an antibody (e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, the delivery vehicle is a liposome according to any of[13]-[72] of the Detailed Description. In some embodiments, theadministered delivery vehicle is pegylated. In some embodiments, theadministered delivery vehicle is not pegylated. In additionalembodiments, the delivery vehicle comprises a targeting moiety on itssurface that has specific affinity for an epitope on an antigen on thesurface of a pancreatic cancer cell. In further embodiments, thedelivery vehicle comprises a targeting moiety that has specific affinityfor an epitope on an antigen selected from TACSTD2 (TROP2), Mucin 1,mesothelin, Guanylyl cyclase C (GCC), SLC44A4, and Nectin 4. In furtherembodiments, the delivery vehicle is a liposome, and the liposomecomprises a targeting moiety has specific affinity for an epitope on anantigen selected from TACSTD2 (TROP2), Mucin 1, Mesothelin, Guanylylcyclase C (GCC), SLC44A4, and Nectin 4. In some embodiments, theadministered delivery vehicle comprises αPANTIFOL containing 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises an alpha tetraglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises an alpha pentaglutamated Antifolate. In other embodiments, theadministered delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises L alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises 2, 3, 4, 5, or more than 5,L-alpha glutamyl groups. In some embodiments, the administered deliveryvehicle comprises D alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, D-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises L and D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, L-alpha glutamyl groups and 2, 3,4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method for treatingbreast cancer (e.g., triple negative breast cancer (estrogen receptor⁻,progesterone receptor⁻, and HER2)) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising alpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosedherein) to a subject having or at risk of having breast cancer. In someembodiments, the delivery vehicle comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the delivery vehicle comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, theadministered delivery vehicle is a liposome that comprises an alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle is a liposome according to any of [13]-[72] of theDetailed Description. In some embodiments, the delivery vehicle is anantibody (e.g., a full-length IgG antibody, a bispecific antibody, or ascFv). In some embodiments, the delivery vehicle is a liposome (e.g., anLp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the administereddelivery vehicle is pegylated. In some embodiments, the administereddelivery vehicle is not pegylated. In additional embodiments, thedelivery vehicle comprises a targeting moiety on its surface that hasspecific affinity for an epitope on an antigen on the surface of abreast cancer cell. In further embodiments, the delivery vehiclecomprises a targeting moiety that has specific affinity for an epitopeon an antigen selected from: LIV-1 (ZIP6), EGFR, HER2, HER3, Mucin 1,GONMB, and Nectin 4. In some embodiments, the targeting moiety is anantibody or a fragment of an antibody. In additional embodiments, thedelivery vehicle is a liposome, and the liposome comprises a targetingmoiety that has specific affinity for an epitope on an antigen selectedfrom: LIV-1 (ZIP6), EGFR, HER2, HER3, Mucin 1, GONMB, and Nectin 4. Insome embodiments, the administered delivery vehicle comprises αPANTIFOLcontaining 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises an alphatetraglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate.

In some embodiments, the disclosure provides a method for treating ahematological cancer that comprises administering an effective amount ofa delivery vehicle (e.g., an antibody or liposome) comprising alphapolyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein) to asubject having or at risk of having a hematological cancer. In someembodiments, the delivery vehicle comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the delivery vehicle comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, thedelivery vehicle is an antibody (e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-αPANTIFOL such as, PLp-αPANTIFOL,NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, the administered delivery vehicle is a liposomeaccording to any of [13]-[72] of the Detailed Description. In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of a hematological cancer cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on an antigen selected from: CD30,CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, CD34, and CD98. Infurther embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety has specific affinity for anepitope on an antigen selected from: CD30, CD79b, CD19, CD138, CD74,CD37, CD19, CD22, CD33, CD34, and CD98. In some embodiments, theadministered delivery vehicle comprises αPANTIFOL containing 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises an alpha tetraglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises an alpha pentaglutamated Antifolate. In other embodiments, theadministered delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises L alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises an alpha pentaglutamatedAntifolate.

In some embodiments, the disclosure provides a method for treating asubject having or at risk of having a cancer that is distinguishable bythe expression of an antigen on its cell surface. Thus, in someembodiments, the disclosure provides a method for treating cancer thatcomprises administering to a subject having or at risk of having acancer, an effective amount of a delivery vehicle (e.g., an antibody orliposome) comprising a targeting moiety that has specific affinity foran epitope on a surface antigen of the cancer and alpha polyglutamatedAntifolate (e.g., an αPANTIFOL disclosed herein). In some embodiments,the delivery vehicle comprises an alpha polyglutamated Antifolateaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the delivery vehicle comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, theadministered delivery vehicle is a liposome according to any of[13]-[72] of the Detailed Description. In some embodiments, theadministered delivery vehicle is pegylated. In some embodiments, thetargeting moiety is an antibody or a fragment of an antibody. Inadditional embodiments, the delivery vehicle is a liposome. In someembodiments, the administered delivery vehicle comprises αPANTIFOLconsisting of 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. Insome embodiments, the administered delivery vehicle comprises an alphatetraglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises 2, 3, 4, 5, ormore than 5, L-alpha glutamyl groups. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered delivery vehicle comprises L and D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In some embodiments, the disclosed compositions (e.g., liposomescontaining alpha polyglutamated Antifolate) are administered to subjectshaving or at risk of having a cancer, a solid tumor, and/or a metastasisthat is distinguishable by the expression of a tumor specific antigen ortumor associated antigen on its cell surface. Thus, in some embodiments,the disclosure provides a method for treating cancer that comprisesadministering an effective amount of a delivery vehicle (e.g., liposome)comprising a targeting moiety and alpha polyglutamated Antifolate (e.g.,an αPANTIFOL disclosed herein) to a subject having or at risk of havinga cancer, solid tumor, and/or metastasis that is distinguishable by theexpression of a tumor specific antigen or tumor associated antigen onits cell surface cancer, and wherein the targeting moiety has specificbinding affinity for an epitope on an tumor specific antigen or tumorassociated antigen. In some embodiments, the delivery vehicle comprisesan alpha polyglutamated Antifolate according to any of [1]-[12] of theDetailed Description. In some embodiments, the delivery vehiclecomprises a polyglutamate of an Antifolate disclosed in Section II,herein. In some embodiments, the administered delivery vehicle is aliposome. In some embodiments, the administered delivery vehicle is aliposome according to any of [53]-[72] of the Detailed Description. Infurther embodiments, the liposome is pegylated. In additionalembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen expressed onthe surface of a cancer, a solid tumor, and/or a metastatic cell. Inadditional embodiments, the targeting moiety has specific affinity foran epitope on an antigen selected from: GONMB, TACSTD2 (TROP2), CEACAM5,EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β orfolate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD40L,CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto,IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4,EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen,Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met,VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO,LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety has specific affinity for anepitope on a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK. In some embodiments, the administered delivery vehicle comprisesαPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the administered delivery vehicle comprisesan alpha tetraglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises an alpha pentaglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises an alpha hexaglutamated Antifolate. In some embodiments, thedelivery vehicle comprises an alpha polyglutamated Antifolate accordingto any of [1]-[12] of the Detailed Description. In some embodiments, thedelivery vehicle comprises a polyglutamate of an Antifolate disclosed inSection II, herein. In other embodiments, the administered deliveryvehicle comprises an alpha hexaglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises L alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, L-alpha glutamylgroups. In some embodiments, the administered delivery vehicle comprisesD alpha polyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises 2, 3, 4, 5, or more than 5, D-alpha glutamylgroups. In some embodiments, the administered delivery vehicle comprisesL and D alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises 2, 3, 4, 5, or more than 5,L-alpha glutamyl groups and 2, 3, 4, 5, or more than 5, D-alpha glutamylgroups. In some embodiments, the administered delivery vehicle is aliposome according to any of [13]-[72] of the Detailed Description.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., an antibody or liposome) comprising a targeting moiety onits surface that specifically binds a folate receptor, and an alphapolyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein) to asubject having or at risk of having a cancer that contains cellsexpressing the folate receptor on their cell surface. In someembodiments, the delivery vehicle comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the delivery vehicle comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, thetargeting moiety is an antibody, or an antigen binding fragment of anantibody. In further embodiments, the targeting moiety has specificaffinity for folate receptor alpha, folate receptor beta or folatereceptor delta. As disclosed herein, the folate receptor targetedpegylated liposomes containing alpha polyglutamated Antifolate are ableto deliver high quantities of alpha polyglutamated Antifolate to cancercells and particularly cancer cells that express folate receptors,compared to normal cells (i.e., cells that unlike cancer cells do notactively take up liposomes, and/or do not express folate receptors). Anycancers that express folate receptors may be treated according to thedisclosed methods. It should be noted that some cancers may expressfolate receptors in an early stage while the majority of cancers mayexpress folate receptors at late stages. In some embodiments, theadministered delivery vehicle is a liposome. In further embodiments, theliposome is pegylated. In some embodiments, the administered deliveryvehicle comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises an alpha tetraglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises an alphapentaglutamated Antifolate. In other embodiments, the administereddelivery vehicle comprises an alpha hexaglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises an alphapentaglutamated Antifolate. In some embodiments, the administereddelivery vehicle is an immunoconjugate. In some embodiments, theadministered delivery vehicle is a liposome. In some embodiments, theadministered delivery vehicle is a liposome according to any of[13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain alphapolyglutamated Antifolate (e.g., an αPANTIFOL disclosed herein) to asubject that is undergoing or has undergone cancer therapy. In someembodiments, administered liposomes comprise an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the administered liposomes comprise an alphapolyglutamated Antifolate that is a polyglutamate of an Antifolatedisclosed in Section II, herein. In some embodiments, the administeredliposomal composition is a PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL. In some embodiments,the administered liposomal composition comprises pegylated liposomes(e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In someembodiments, the administered liposomal composition comprises atargeting moiety that has specific affinity for an epitope on a surfaceantigen of a cancer cell (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). Insome embodiments, the administered liposomal composition comprisesliposomes that are pegylated and comprise a targeting moiety (e.g.,TPLp-αPANTIFOL). In some embodiments, the administered liposomalcomposition comprises liposomes that are targeted and liposomes that donot comprise a targeting moiety (e.g., are not targeted). In someembodiments, the administered liposomal composition comprises liposomesthat are pegylated and liposomes that are not pegylated. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha polyglutamated Antifolate that contains 4, 5, 6, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, liposomes ofthe administered liposomal composition comprise alpha tetraglutamatedAntifolate. In some embodiments, liposomes of the administered liposomalcomposition comprise alpha pentaglutamated Antifolate. In otherembodiments, liposomes of the administered liposomal compositioncomprise alpha hexaglutamated Antifolate.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is a solid tumor lymphoma. Examples of solid tumorlymphoma include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B celllymphoma.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is bone cancer, brain cancer, breast cancer, colorectalcancer, connective tissue cancer, cancer of the digestive system,endometrial cancer, esophageal cancer, eye cancer, cancer of the headand neck, gastric cancer, intra-epithelial neoplasm, melanomaneuroblastoma, Non-Hodgkin's lymphoma, non-small cell lung cancer,prostate cancer, retinoblastoma, or rhabdomyosarcoma.

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a compositioncomprising a delivery vehicle and alpha polyglutamated Antifolate to asubject having or at risk of having cancer. In some embodiments, theadministered composition comprises a pegylated delivery vehicle. In someembodiments, the administered composition comprises a targeting moietythat has a specific affinity for an epitope of antigen on the surface ofa target cell of interest such as a cancer cell. In some embodiments,the delivery vehicle comprises an antibody or an antigen bindingantibody fragment. In some embodiments, the composition is administeredto treat a cancer selected from: lung cancer, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, head and neck cancer, gastriccancer, gastrointestinal cancer, colorectal cancer, esophageal cancer,cervical cancer, liver cancer, kidney cancer, biliary duct cancer,gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), braincancer, central nervous system cancer, melanoma, myeloma, a leukemia anda lymphoma. In some embodiments, the composition is administered totreat a cancer selected from: breast cancer, advanced head and neckcancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin'slymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, chorioadenoma, nonleukemicmeningeal cancer, soft tissue sarcoma (desmoid tumors, aggressivefibromatosis), bladder cancer, and central nervous system (CNS). In someembodiments, the treated cancer is a metastasis of one of the abovelisted cancers. In some embodiments, the administered compositioncontains 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises an alphatetraglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle is an immunoconjugate. In some embodiments, theadministered delivery vehicle comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the liposomal composition comprises a polyglutamate ofan Antifolate disclosed in Section II, herein. In some embodiments, theadministered delivery vehicle is a liposome. In some embodiments, theadministered delivery vehicle is a liposome according to any of[13]-[72] of the Detailed Description.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition comprising liposomes that contain alpha polyglutamatedAntifolate (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL,NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) to a subject having orat risk of having cancer. In some embodiments, the liposomal compositioncomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomalcomposition comprises a polyglutamate of an Antifolate disclosed inSection II, herein. In some embodiments, the liposomal compositioncomprises a liposome according to any of [13]-[72] of the DetailedDescription. In some embodiments, the liposomal composition isadministered to treat a cancer selected from: lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments,liposomes of the administered liposomal composition comprise a αPANTIFOLcontaining 4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha tetraglutamated Antifolate. In some embodiments,liposomes of the administered liposomal composition comprise alphapentaglutamated Antifolate. In other embodiments, liposomes of theadministered liposomal composition comprises an alpha hexaglutamatedAntifolate.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that comprises targeted liposomes (e.g., TLp-αPANTIFOL orTPLp-αPANTIFOL) to a subject having or at risk of having cancer, whereinthe liposomal composition comprises liposomes that comprise alphapolyglutamated Antifolate (Lp-αPANTIFOL) and further comprise atargeting moiety having a specific affinity for a surface antigen(epitope) on the cancer. In some embodiments, the liposomal compositioncomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomalcomposition comprises a polyglutamate of an Antifolate disclosed inSection II, herein. In some embodiments, the liposomal compositioncomprises a liposome according to any of [53]-[72] of the DetailedDescription. In some embodiments, the liposomal composition isadministered to treat a cancer selected from: lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the liposomalcomposition is administered to treat a cancer selected from: breastcancer, advanced head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis), bladder cancer, and centralnervous system (CNS) cancer.

In some embodiments, the cancer is lung cancer (e.g., NSCLC ormesothelioma). In some embodiments, the cancer is breast cancer (e.g.,HER2++ or triple negative breast cancer). In some embodiments, thecancer is colorectal cancer. In some embodiments, the cancer is ovariancancer. In some embodiments, the cancer is endometrial cancer. In someembodiments, the cancer is pancreatic cancer. In some embodiments, thecancer is liver cancer. In some embodiments, the cancer is head and neckcancer. In some embodiments, the cancer is osteosarcoma. In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, liposomes of the administered liposomal compositioncomprise an αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, liposomes of the administeredliposomal composition comprise alpha tetraglutamated Antifolate. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha pentaglutamated Antifolate. In other embodiments,liposomes of the administered liposomal composition comprises an alphahexaglutamated Antifolate. In some embodiments, the administeredliposomal composition comprises L alpha polyglutamated Antifolate. Insome embodiments, the administered liposomal composition comprises 2, 3,4, 5, or more than 5, L-alpha glutamyl groups. In some embodiments, theadministered liposomal composition comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered liposomal compositioncomprises 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups. In someembodiments, the administered liposomal composition comprises L and Dalpha polyglutamated Antifolate. In some embodiments, the administeredliposomal composition comprises 2, 3, 4, 5, or more than 5, L-alphaglutamyl groups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that comprises targeted liposomes (e.g., TLp-αPANTIFOL orTPLp-αPANTIFOL) to a subject having or at risk of having cancer, whereinthe liposomal composition comprises liposomes that comprise alphapolyglutamated Antifolate (Lp-αPANTIFOL) and further comprise atargeting moiety having a specific affinity for a surface antigen(epitope) on the cancer. In some embodiments, the liposomal compositioncomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomalcomposition comprises a polyglutamate of an Antifolate disclosed inSection II, herein. In some embodiments, the targeted liposome is aliposome according to any of [53]-[72] of the Detailed Description. Insome embodiments, the liposomal composition is administered to treat acancer selected from: lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma. In some embodiments, the liposomal composition is administeredto treat a cancer selected from: breast cancer, advanced head and neckcancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin'slymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, chorioadenoma, nonleukemicmeningeal cancer, soft tissue sarcoma (desmoid tumors, aggressivefibromatosis), bladder cancer, and central nervous system (CNS) cancer.In some embodiments, the liposomal composition is administered to treatlung cancer (e.g., NSCLC or mesothelioma). In some embodiments, theliposomal composition is administered to treat breast cancer (e.g.,HER2++ or triple negative breast cancer). In some embodiments, theliposomal composition is administered to treat colorectal cancer. Insome embodiments, the liposomal composition is administered to treatovarian cancer. In some embodiments, the liposomal composition isadministered to treat endometrial cancer. In some embodiments, theliposomal composition is administered to treat pancreatic cancer. Insome embodiments, the liposomal composition is administered to treatliver cancer. In some embodiments, the liposomal composition isadministered to treat head and neck cancer. In some embodiments, theliposomal composition is administered to treat osteosarcoma. In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). Insome embodiments, liposomes of the administered liposomal compositioncomprise an αPANTIFOL containing 4, 5, 6, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, liposomes of the administeredliposomal composition comprise alpha tetraglutamated Antifolate. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha pentaglutamated Antifolate. In other embodiments,liposomes of the administered liposomal composition comprises an alphahexaglutamated Antifolate. In some embodiments, the administeredliposomal composition comprises L alpha polyglutamated Antifolate. Insome embodiments, the administered liposomal composition comprises Dalpha polyglutamated Antifolate. In some embodiments, the administeredliposomal composition comprises L and D alpha polyglutamated Antifolate.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that contains targeted liposomes (e.g., TLp-αPANTIFOL orTPLp-αPANTIFOL) to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) alpha polyglutamatedAntifolate (αPANTIFOL) and (b) a targeting moiety that has specificbinding affinity for the folate receptor. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,TPLp-αPANTIFOL). In some embodiments, the liposomal compositioncomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Detailed Description. In some embodiments, the liposomalcomposition comprises a polyglutamate of an Antifolate disclosed inSection II, herein. In some embodiments, the liposomal compositioncomprises a liposome is a liposome according to any of [53]-[72] of theDetailed Description. In some embodiments, the targeting moiety has aspecific binding affinity for folate receptor alpha (FR-α), folatereceptor beta (FR-β), and/or folate receptor delta (FR-δ). In someembodiments, the targeting moiety has a specific binding affinity forfolate receptor alpha (FR-α), folate receptor beta (FR-β), and/or folatereceptor delta (FR-α). In some embodiments, the targeting moiety has aspecific binding affinity for folate receptor alpha (FR-α) and folatereceptor beta (FR-β), and/or folate receptor delta (FR-δ). In someembodiments, the targeting moiety has a specific binding affinity forfolate receptor alpha (FR-α) and folate receptor beta (FR-β), and/orfolate receptor delta (FR-δ). In some embodiments, the targeting moietyhas a specific binding affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In some embodiments, the liposomalcomposition is administered to treat a cancer selected from: lungcancer, pancreatic, breast cancer, ovarian cancer, lung cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colon cancer, esophageal cancer, cervical cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, and a hematologic malignancy. In someembodiments, the liposomal composition is administered to treat a cancerselected from: breast cancer, advanced head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis), bladdercancer, and central nervous system (CNS) cancer. In some embodiments,the liposomal composition is administered to treat lung cancer (e.g.,NSCLC or mesothelioma). In some embodiments, the liposomal compositionis administered to treat breast cancer (e.g., HER2++ or triple negativebreast cancer). In some embodiments, the liposomal composition isadministered to treat colorectal cancer. In some embodiments, theliposomal composition is administered to treat ovarian cancer. In someembodiments, the liposomal composition is administered to treatendometrial cancer. In some embodiments, the liposomal composition isadministered to treat pancreatic cancer. In some embodiments, theliposomal composition is administered to treat liver cancer. In someembodiments, the liposomal composition is administered to treat head andneck cancer. In some embodiments, the liposomal composition isadministered to treat osteosarcoma. In some embodiments, liposomes ofthe administered liposomal composition comprise an αPANTIFOL containing4, 5, 6, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, liposomes of the administered liposomal compositioncomprise alpha tetraglutamated Antifolate. In some embodiments,liposomes of the administered liposomal composition comprise alphapentaglutamated Antifolate. In other embodiments, liposomes of theadministered liposomal composition comprises an alpha hexaglutamatedAntifolate. In some embodiments, the administered liposomal compositioncomprises L alpha polyglutamated Antifolate. In some embodiments, theadministered liposomal composition comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered liposomal compositioncomprises L and D alpha polyglutamated Antifolate.

In some embodiments, the disclosure provides a method for treating adisorder of the immune system (e.g., an autoimmune disease such asinflammation and rheumatoid arthritis) that comprises administering aneffective amount of a delivery vehicle (e.g., antibody or liposome)comprising alpha polyglutamated Antifolate (e.g., an αPANTIFOL disclosedherein) to a subject having or at risk of having a disorder of theimmune system. In some embodiments, the delivery vehicle comprises analpha polyglutamated Antifolate according to any of [1]-[12] of theDetailed Description. In some embodiments, the delivery vehiclecomprises a polyglutamate of an Antifolate disclosed in Section II,herein. In some embodiments, the delivery vehicle is an antibody (e.g.,a full-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-αPANTIFOLsuch as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL,or TPLp-αPANTIFOL). In some embodiments, the delivery vehicle is aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of an immune cell associated with a disorder of theimmune system. In some embodiments, the targeting moiety is an antibodyor an antigen binding antibody fragment. In some embodiments, theadministered delivery vehicle comprises αPANTIFOL containing 4, 5, 6,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises an alpha tetraglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises an alpha pentaglutamated Antifolate. In other embodiments, theadministered delivery vehicle comprises an alpha hexaglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises L alpha polyglutamated Antifolate. In some embodiments, theadministered delivery vehicle comprises D alpha polyglutamatedAntifolate. In some embodiments, the administered delivery vehiclecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the autoimmune disease is rheumatoid arthritis.

In some embodiments, the disclosure provides a method for treating aninfectious disease (e.g., HIV, malaria, and schistomiasis) thatcomprises administering an effective amount of a delivery vehicle (e.g.,antibody or liposome) comprising alpha polyglutamated Antifolate (e.g.,an αPANTIFOL disclosed herein) to a subject having or at risk of havingan infectious disease. In some embodiments, the delivery vehiclecomprises an alpha polyglutamated Antifolate according to any of[1]-[12] of the Detailed Description. In some embodiments, the deliveryvehicle comprises a polyglutamate of an Antifolate disclosed in SectionII, herein. In some embodiments, the delivery vehicle is an antibody(e.g., a full-length IgG antibody, a bispecific antibody, or a scFv). Insome embodiments, the delivery vehicle is a liposome (e.g., anLp-αPANTIFOL such as, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL,TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the deliveryvehicle is a liposome according to any of [13]-[72] of the DetailedDescription. In some embodiments, the administered delivery vehicle ispegylated. In some embodiments, the administered delivery vehicle is notpegylated. In additional embodiments, the administered delivery vehiclecomprises a targeting moiety that has a specific affinity for an epitopeof antigen on the surface of a pathogen associated with an infectiousdisease. In some embodiments, the targeting moiety is an antibody or anantigen binding antibody fragment. In some embodiments, the administereddelivery vehicle comprises αPANTIFOL containing 4, 5, 6, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the administereddelivery vehicle comprises an alpha pentaglutamated Antifolate. In otherembodiments, the administered delivery vehicle comprises an alphahexaglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L alpha polyglutamated Antifolate. In someembodiments, the administered delivery vehicle comprises D alphapolyglutamated Antifolate. In some embodiments, the administereddelivery vehicle comprises L and D alpha polyglutamated Antifolate.

In some embodiments, the administered delivery vehicle is a liposome. Insome embodiments, the liposome comprises an alpha polyglutamatedAntifolate according to any of [1]-[12] of the Detailed Description. Insome embodiments, the liposome comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, thedelivery vehicle is a liposome according to any of [13]-[72] of theDetailed Description. In further embodiments, the liposome is pegylated.In additional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope on thesurface of a target cell of interest. In some embodiments, the deliveryvehicle is a targeted liposome according to any of [13]-[72] of theDetailed Description. In further embodiments, the delivery vehiclecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety that has specific affinity for anepitope on a cell surface antigen selected from: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133,CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2,EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), aC242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg,CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA,TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, andMuSK. In some embodiments, the delivery vehicle comprises an alphapolyglutamated Antifolate according to any of [1]-[12] of the DetailedDescription. In some embodiments, the liposome comprises a polyglutamateof an Antifolate disclosed in Section II, herein. In some embodiments,the delivery vehicle is a targeted liposome according to any of[53]-[72] of the Detailed Description.

In some embodiments, the disclosure provides for the use of acomposition comprising an alpha polyglutamated Antifolate formanufacture of a medicament for treatment of a hyperproliferativedisease. In some embodiments, the composition comprises an alphapolyglutamated Antifolate according to any of [1]-[12] of the DetailedDescription. In some embodiments, the composition comprises apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the composition comprises a liposome according to any of[13]-[72] of the Detailed Description. In some embodiments, thecomposition comprises a targeted liposome according to any of [53]-[72]of the Detailed Description that comprises a targeting moiety. In someembodiments, the alpha polyglutamated Antifolate comprise 5 or moreglutamyl groups. In some embodiments, the alpha polyglutamatedAntifolate is tetraglutamated. In some embodiments, the alphapolyglutamated Antifolate is pentaglutamated or hexaglutamated. In someembodiments, the alpha polyglutamated Antifolate is pentaglutamated. Insome embodiments, the alpha polyglutamated Antifolate is hexaglutamated.In some embodiments, the alpha polyglutamated Antifolate is in aliposome. In some embodiments, the hyperproliferative disease is aneoplasia, tumor, or cancer, or metastasis thereof. In some embodiments,the hyperproliferative disease is cancer. In some embodiments, thecancer is selected from: lung (e.g., non-small lung cancer), pancreatic,breast cancer, ovarian, lung, prostate, head and neck, gastric,gastrointestinal, colon, esophageal, cervical, kidney, biliary duct,gallbladder, and a hematologic malignancy. In some embodiments, thecancer is selected from: breast cancer, advanced head and neck cancer,lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis), bladdercancer, and central nervous system (CNS) cancer. In some embodiments,the cancer is lung cancer (e.g., NSCLC or mesothelioma). In someembodiments, the cancer is breast cancer (e.g., HER2++ or triplenegative breast cancer). In some embodiments, the cancer is colorectalcancer. In some embodiments, the cancer is ovarian cancer. In someembodiments, the cancer is endometrial cancer. In some embodiments, thecancer is pancreatic cancer. In some embodiments, the cancer is livercancer. In some embodiments, the cancer is head and neck cancer. In someembodiments, the cancer is osteosarcoma. In some embodiments, the canceris leukemia or lymphoma. In some embodiments, the hyperproliferativedisease is an autoimmune disease. In some embodiments, thehyperproliferative disease is rheumatoid arthritis.

The disclosed methods can be practiced in any subject that is likely tobenefit from delivery of compositions contemplated herein (e.g., alphapolyglutamated Antifolate compositions such as liposome containing apentaglutamated or hexaglutamated Antifolate). Mammalian subjects, andin particular, human subjects are preferred. In some embodiments, thesubjects also include animals such as household pets (e.g., dogs, cats,rabbits, and ferrets), livestock or farm animals (e.g., cows, pigs,sheep, chickens and other poultry), horses such as thoroughbred horses,laboratory animals (e.g., mice, rats, and rabbits), and other mammals.In other embodiments, the subjects include fish and other aquaticspecies.

The subjects to whom the agents are delivered may be normal subjects.Alternatively, the subject may have or be at risk of developing acondition that can be diagnosed or that can benefit from delivery of oneor more of the provided compositions. In some embodiments, suchconditions include cancer (e.g., solid tumor cancers or non-solid cancersuch as leukemias). In some embodiments, these conditions (e.g.,cancers) involve cells that express an antigen that can be specificallybound by a targeted pegylated liposomal alpha polyglutamated Antifolatedisclosed herein. In further embodiments, these antigens specificallybind and internalize the targeted pegylated liposomal alphapolyglutamated Antifolate into the cell. In some embodiments, thetargeted pegylated liposomal alpha polyglutamated Antifolatespecifically binds a folate receptor (e.g., folate receptor alpha(FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ))expressed on the surface of the cancer cell.

Tests for diagnosing the conditions that can be treated with theprovided compositions are known in the art and will be familiar to themedical practitioner. The determination of whether a cell type expressesfolate receptors can be made using commercially available antibodies.These laboratory tests include without limitation microscopic analyses,cultivation dependent tests (such as cultures), and nucleic aciddetection tests. These include wet mounts, stain-enhanced microscopy,immune microscopy (e.g., FISH), hybridization microscopy, particleagglutination, enzyme-linked immunosorbent assays, urine screeningtests, DNA probe hybridization, and serologic tests. The medicalpractitioner will generally also take a full history and conduct acomplete physical examination in addition to running the laboratorytests listed above.

A subject having a cancer can, for example, be a subject that hasdetectable cancer cells. A subject at risk of developing a cancer can,for example, be a subject that has a higher than normal probability ofdeveloping cancer. These subjects include, for instance, subjects havinga genetic abnormality that has been demonstrated to be associated with ahigher likelihood of developing a cancer, subjects having a familialdisposition to cancer, subjects exposed to cancer causing agents (i.e.,carcinogens) such as tobacco, asbestos, or other chemical toxins, andsubjects previously treated for cancer and in apparent remission.

In some embodiments, the disclosure provides methods for selectivelydeliver a folate receptor targeted pegylated liposomal alphapolyglutamated Antifolate to a tumor cell expressing a folate receptoron its surface at a rate that is higher (e.g., at least two-foldgreater, at least three-fold greater, at least four-fold greater, or atleast five-fold greater, than a cell not expressing folate receptor onits cell surface). In some embodiments, the delivered pegylated liposomecomprises an alpha polyglutamated Antifolate. In some embodiments, thepegylated liposome comprises an alpha polyglutamated Antifolateaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the pegylated liposome comprises a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, theliposome is a liposome according to any of [53]-[72] of the DetailedDescription. In some embodiments, the pegylated liposome comprises 2, 3,4, 5, or more than 5, L-alpha glutamyl groups. In some embodiments, thepegylated liposome comprises D alpha polyglutamated Antifolate. In someembodiments, the pegylated liposome comprises 2, 3, 4, 5, or more than5, D-alpha glutamyl groups. In some embodiments, the pegylated liposomecomprises L and D alpha polyglutamated Antifolate. In some embodiments,the pegylated liposome comprises 2, 3, 4, 5, or more than 5, L-alphaglutamyl groups and 2, 3, 4, 5, or more than 5, D-alpha glutamyl groups.

V. Combination Therapy

In certain embodiments, in addition to administering alphapolyglutamated Antifolate composition described herein, the method ortreatment further comprises administering at least one additionaltherapeutic agent. An additional therapeutic agent can be administeredprior to, concurrently with, and/or subsequently to, administration ofthe alpha polyglutamated Antifolate composition. In some embodiments,the administered alpha polyglutamated Antifolate is a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the administered alpha polyglutamated Antifolate is apolyglutamate of an Antifolate disclosed in Section II, herein. Theadditional therapeutic agent can be associated with an alphapolyglutamated Antifolate delivery vehicle (e.g., coencapsulated withalpha polyglutamated Antifolate in a liposome), present in a solutioncontaining an alpha polyglutamated Antifolate delivery vehicle, or in aseparate formulation from the composition containing the alphapolyglutamated Antifolate composition. Pharmaceutical compositionscomprising a polypeptide or agent and the additional therapeuticagent(s) are also provided. In some embodiments, the at least oneadditional therapeutic agent comprises 1, 2, 3, or more additionaltherapeutic agents.

Combination therapy with two or more therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of the polypeptide or agent(s). Combination therapy may decreasethe likelihood that resistant cancer cells will develop. In someembodiments, combination therapy comprises a therapeutic agent thataffects the immune response (e.g., enhances or activates the response)and a therapeutic agent that affects (e.g., inhibits or kills) thetumor/cancer cells.

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of an alphapolyglutamated Antifolate composition disclosed herein and a biologic.In some embodiments, the administered alpha polyglutamated Antifolate isa αPANTFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the administered alpha polyglutamated Antifolate is apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the administered alpha polyglutamated Antifolate is in aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the alpha polyglutamated Antifolate is administered incombination with a therapeutic antibody. In further embodiments, thealpha polyglutamated Antifolate is administered in combination with ananti-CD antibody (e.g., rituximab) or an antibody that binds an immunecheckpoint protein (e.g., CTLA4, PD1, PDL1, and TIM3). In furtherembodiments, the alpha polyglutamated Antifolate is administered incombination with an fc-fusion protein (e.g., entanercept).

In some embodiments, the disclosure provides a method for treatingdisorder of the immune system that comprises administering an effectiveamount of an alpha polyglutamated Antifolate composition disclosedherein and a biologic. In some embodiments, the administered alphapolyglutamated Antifolate is an αPANTIFOL according to any of [1]-[12]of the Detailed Description. In some embodiments, the administered alphapolyglutamated Antifolate is a polyglutamate of an Antifolate disclosedin Section II, herein. In some embodiments, the administered alphapolyglutamated Antifolate is in a liposome according to any of [13]-[72]of the Detailed Description. In some embodiments, the alphapolyglutamated Antifolate is administered in combination with atherapeutic antibody. In further embodiments, the alpha polyglutamatedAntifolate is administered in combination with an anti-TNF antibody(e.g., adalimumab). In some embodiments, the alpha polyglutamatedAntifolate is administered in combination with an fc-fusion protein(e.g., entanercept).

In some embodiments, of the methods described herein, the combination ofan αPANTIFOL compositions described herein and at least one additionaltherapeutic agent results in additive or synergistic results. In someembodiments, the combination therapy results in an increase in thetherapeutic index of the αPANTIFOL or agent. In some embodiments, thecombination therapy results in an increase in the therapeutic index ofthe additional therapeutic agent(s). In some embodiments, thecombination therapy results in a decrease in the toxicity and/or sideeffects of the αPANTIFOL or agent. In some embodiments, the combinationtherapy results in a decrease in the toxicity and/or side effects of theadditional therapeutic agent(s).

In some embodiments, in addition to administering alpha polyglutamatedAntifolate compositions described herein, the methods or treatmentsdescribed herein further comprise administering at least one additionaltherapeutic agent selected from: an anti-tubulin agent, an auristatin, aDNA minor groove binder, a DNA replication inhibitor, an alkylatingagent (e.g., platinum complexes such as cisplatin, mono(platinum),bis(platinum) and tri-nuclear platinum complexes and carboplatin), ananthracycline, an antibiotic, an anti-folate (e.g., a polyglutamatableantifolate or a non polyglutamatable anti-folate), an antimitotic (e.g.,a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, orvindesine), radiation sensitizer, a steroid, a taxane, a topoisomeraseinhibitor (e.g., doxorubicin HCl, daunorubicin citrate, mitoxantroneHCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), andirinotecan), an anti-metabolite, a chemotherapy sensitizer, aduocarmycin, an etoposide, a fluorinated pyrimidine, an ionophore, alexitropsin, a nitrosourea, a platinol, a purine antimetabolite, a PARPinhibitor, and a puromycin. In certain embodiments, the secondtherapeutic agent is an alkylating agent, an antimetabolite, anantimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor. Insome embodiments, the administered alpha polyglutamated Antifolate is aαPANTIFOL according to any of [1]-[12] of the Detailed Description. Insome embodiments, the administered alpha polyglutamated Antifolate is apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the administered alpha polyglutamated Antifolate is in aliposome according to any of [13]-[72] of the Detailed Description.

Therapeutic agents that may be administered in combination with theαPANTIFOL compositions described herein include chemotherapeutic agents.Thus, in some embodiments, the methods or treatments described hereinfurther comprise administering at least one involves the administrationof a αPANTIFOL composition described herein in combination with achemotherapeutic agent or in combination with a cocktail ofchemotherapeutic agents. Treatment with a αPANTIFOL composition canoccur prior to, concurrently with, or subsequent to administration ofchemotherapies. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in The Chemotherapy Source Book, 4th Edition, 2008, M. C.Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the present invention include, but arenot limited to, alkylating agents such as thiotepa and cyclophosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as Antifolate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,Antifolate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, such as paclitaxel (TAXOL®) and docetaxel(TAXOTERE®); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA®); anti-hormonal agents such as, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene(FARESTON®); anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, theadditional therapeutic agent is cisplatin. In certain embodiments, theadditional therapeutic agent is carboplatin. In other embodiments, theadditional therapeutic agent is oxaloplatin.

Additional therapeutic agents that may be administered in combinationwith the αPANTIFOL compositions described herein include one or moreimmunotherapeutic agents.

In some embodiments an αPANTIFOL composition described herein isadministered in combination with an immunotherapeutic agent thatinhibits one or more T cell-associated inhibitory molecules (e.g.,CTLA4, PD1, Lymphocyte activation gene-3 (LAG-3, CD223), T cellimmunoglobulin-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT),V-domain Ig suppressor of T cell activation (VISTA), B7 homolog 3(B7-H3, CD276), B and T cell lymphocyte attenuator (BTLA, CD272), orAdenosine A2a receptor (A2aR) or CD73). In some embodiments, theαPANTIFOL composition is administered separately from theimmunotherapeutic agent. In some embodiments, the αPANTIFOL compositionis administered at the same time (e.g., concurrently or serially) as theimmunotherapeutic agent. In some embodiments, the αPANTIFOL compositionand the immunotherapeutic agent are encapsulated in or otherwiseassociated with the same liposome.

In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith a PD1 inhibitor. In some embodiments, the αPANTIFOL composition isadministered in combination with pembroluzumab. In some embodiments, theαPANTIFOL composition is administered in combination with nivolumab. Insome embodiments, the αPANTIFOL composition is administered separatelyfrom the PD1 inhibitor. In some embodiments, the αPANTIFOL compositionis administered at the same time (e.g., concurrently or serially) as thePD1 inhibitor. In some embodiments, the αPANTIFOL composition and thePD1 inhibitor are encapsulated in or otherwise associated with the sameliposome.

In other embodiments, the αPANTIFOL composition is administered incombination with a PDL1 inhibitor. In some embodiments, t the αPANTIFOLcomposition is administered in combination with atezolizumab. In someembodiments, the αPANTIFOL composition is administered in combinationwith avelumab. In some embodiments, the αPANTIFOL composition isadministered in combination with durvalumab. In some embodiments, theαPANTIFOL composition is administered in combination with PDR001. Insome embodiments, the αPANTIFOL composition is administered separatelyfrom the PDL-1 inhibitor. In some embodiments, the αPANTIFOL compositionis administered at the same time (e.g., concurrently or serially) as thePDL-1 inhibitor. In some embodiments, the αPANTIFOL composition and thePDL-1 inhibitor are encapsulated in or otherwise associated with thesame liposome.

In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition in combination with a therapeuticagent that inhibits activity of CTLA4 LAG3, TIM-3, TIGIT, VISTA, B7-H3,BTLA, A2aR or CD73. In some embodiments, treatment methods providedherein comprise administering an αPANTIFOL composition described hereinin combination with a CTLA4 inhibitor. In further embodiments, theαPANTIFOL composition is administered in combination with ipilimumab. Insome embodiments, treatment methods provided herein compriseadministering a αPANTIFOL composition in combination with a LAG3inhibitor. In further embodiments, the αPANTIFOL composition isadministered in combination with TSR-033, MK-4280, LAG525, BMS-986106,or MGD013. In some embodiments, treatment methods provided hereincomprise administering a αPANTIFOL composition in combination with aTIM-3 inhibitor. In further embodiments, the αPANTIFOL composition isadministered in combination with MBG453 or MEDI9447. In someembodiments, treatment methods provided herein comprise administering aαPANTIFOL composition in combination with a TIGIT inhibitor. In furtherembodiments, an αPANTIFOL composition is administered in combinationwith BMS-986207 or OMP-31M32. In some embodiments, treatment methodsprovided herein comprise administering the αPANTIFOL composition incombination with a VISTA inhibitor. In further embodiments, theαPANTIFOL composition is administered in combination with JNJ-61610588or CA-170. In some embodiments, treatment methods provided hereincomprise administering a αPANTIFOL composition in combination with aB7-H3 inhibitor. In further embodiments, the αPANTIFOL composition isadministered in combination with neoblituzumab, enoblituzumab, MGD009,or 8H9. In some embodiments, treatment methods provided herein compriseadministering a αPANTIFOL composition in combination with a BTLAinhibitor. In some embodiments, treatment methods provided hereincomprise administering a αPANTIFOL composition in combination with anA2aR or CD73 inhibitor. In further embodiments, the αPANTIFOLcomposition is administered in combination with CPI444. In someembodiments, the αPANTIFOL composition is administered separately fromthe immunotherapeutic agent. In some embodiments, the αPANTIFOLcomposition is administered at the same time (e.g., concurrently orserially) as the immunotherapeutic agent. In some embodiments, theαPANTIFOL composition and the immunotherapeutic agent are encapsulatedin or otherwise associated with the same liposome.

In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition in combination with a therapeuticagent that inhibits activity of transforming growth factor (TGF)-β,phosphoinositide 3-kinase gamma (PI3Kγ), Killer immunoglobulin-likereceptors (KIR, CD158), CD47, or Indoleamine 2,3-dioxygenase (IDO). Insome embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith a TGFβ antagonist. In further embodiments, the αPANTIFOLcomposition is administered in combination with M7824 or Galusertinib(LY2157299). In some embodiments, treatment methods provided hereincomprise administering an αPANTIFOL composition described herein incombination with a PI3Kγ antagonist. In further embodiments, theαPANTIFOL composition is administered in combination with IPI-549. Insome embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith a KIR antagonist. In further embodiments, the αPANTIFOL compositionis administered in combination with IPH4102 or lirilumab. In someembodiments, treatment methods provided herein comprise administering anαPANTIFOL composition described herein in combination with a CD47antagonist. In further embodiments, the αPANTIFOL composition isadministered in combination with Hu5F9-G4 or TTI-621. In someembodiments, treatment methods provided herein comprise administering anαPANTIFOL composition described herein in combination with an IDOantagonist. In further embodiments, the αPANTIFOL composition isadministered in combination with BMS-986205, indoximod, or epacadostat.In some embodiments, the αPANTIFOL composition is administeredseparately from the therapeutic agent. In some embodiments, theαPANTIFOL composition is administered at the same time (e.g.,concurrently or serially) as the therapeutic agent. In some embodiments,the αPANTIFOL composition and the therapeutic agent are encapsulated inor otherwise associated with the same liposome.

In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition in combination with a therapeuticagent that is an agonist of OX40 (CD134), inducible co-stimulator(ICOS), Glucocorticoid-induced TNF receptor family-related protein(GITR), 4-1BB (CD137), CD40, CD27-CD70, or a Toll-like receptor (TLR).In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith an OX40 agonist. In further embodiments, the αPANTIFOL compositionis administered in combination with GSK3174998, MOXR0916, 9B12,PF-04518600 (PF-8600), MEDI6383, MEDI0562, INCAGN01949, or GSK3174998.In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith an ICOS agonist. In further embodiments, the αPANTIFOL compositionis administered in combination with JTX-2011, GSK3359609, or MEDI-570.In some embodiments, treatment methods provided herein compriseadministering an αPANTIFOL composition described herein in combinationwith a GITR agonist. In further embodiments, the αPANTIFOL compositionis administered in combination with TRX-518, AMG 228, BMS-986156,MEDI1873, MK-4166, INCAGN01876, or GWN32. In some embodiments, treatmentmethods provided herein comprise administering an αPANTIFOL compositiondescribed herein in combination with a 4-1BB agonist. In furtherembodiments, the αPANTIFOL composition is administered in combinationwith utomilumab or urelumab (PF-05082566). In some embodiments,treatment methods provided herein comprise administering an αPANTIFOLcomposition described herein in combination with a CD40 agonist. Infurther embodiments, the αPANTIFOL composition is administered incombination with CP-870893, APX005M, ADC-1013, lucatumumab, Chi Lob 7/4,dacetuzumab, SEA-CD40, or RO7009789. In some embodiments, treatmentmethods provided herein comprise administering an αPANTIFOL compositiondescribed herein in combination with a CD27-CD70 agonist. In furtherembodiments, the αPANTIFOL composition is administered in combinationwith ARGX-110, or BMS-936561 (MDX-1203). In some embodiments, treatmentmethods provided herein comprise administering an αPANTIFOL compositiondescribed herein in combination with a TLR agonist. In furtherembodiments, the αPANTIFOL composition is administered in combinationwith MEDI9197, PG545 (pixatimod, pINN), or poly-ICLC. In someembodiments, the αPANTIFOL composition is administered separately fromthe therapeutic agent. In some embodiments, the αPANTIFOL composition isadministered at the same time (e.g., concurrently or serially) as thetherapeutic agent. In some embodiments, the αPANTIFOL composition andthe therapeutic agent are encapsulated in or otherwise associated withthe same liposome.

In some embodiments, the disclosure provides a combination therapywherein an alpha polyglutamated Antifolate composition described hereinis administered in combination a DMARD. In some embodiments, theadministered alpha polyglutamated Antifolate is an αPANTIFOL accordingto any of [1]-[12] of the Detailed Description. In some embodiments, theadministered alpha polyglutamated Antifolate is a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, theadministered alpha polyglutamated Antifolate is in a liposome accordingto any of [13]-[72] of the Detailed Description. In further embodiments,the alpha polyglutamated Antifolate composition is administered incombination sulfasalazine or hydroxychloroquine. In some embodiments,the disclosure provides a combination therapy wherein a gammapolyglutamated Antifolate composition described herein is administeredin combination with chloroquine.

In some embodiments, the disclosure provides a combination therapywherein an alpha polyglutamated Antifolate composition described hereinis administered in combination with a steroid. In some embodiments, theadministered alpha polyglutamated Antifolate is a αPANTIFOL according toany of [1]-[12] of the Detailed Description. In some embodiments, theadministered alpha polyglutamated Antifolate is a polyglutamate of anAntifolate disclosed in Section II, herein. In some embodiments, theadministered alpha polyglutamated Antifolate is in a liposome accordingto any of [13]-[72] of the Detailed Description. In further embodiments,the alpha polyglutamated Antifolate composition is administered incombination with prednisolone.

In some embodiments, the disclosure provides a combination therapywherein an alpha polyglutamated Antifolate composition described hereinis administered in combination a biologic agent. In some embodiments,the administered alpha polyglutamated Antifolate is a αPANTIFOLaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the administered alpha polyglutamated Antifolate is apolyglutamate of an Antifolate disclosed in Section II, herein. In someembodiments, the administered alpha polyglutamated Antifolate is in aliposome according to any of [13]-[72] of the Detailed Description. Insome embodiments, the biologic agent is a therapeutic antibody. Infurther embodiments, the therapeutic binds TNF-alpha or CD-20.

VI. Kits Comprising αPANTIFOL Compositions

The disclosure also provides kits that comprise the αPANTIFOLcompositions described herein and that can be used to perform themethods described herein. In certain embodiments, a kit comprises atleast one purified αPANTIFOL composition in one or more containers. Insome embodiments, the kit comprises an alpha polyglutamated Antifolateaccording to any of [1]-[12] of the Detailed Description. In someembodiments, the kit comprises an alpha polyglutamated Antifolatewherein the Antifolate is a polyglutamate of an Antifolate disclosed inSection II, herein. In some embodiments, the kit comprises an alphapolyglutamated Antifolate according to any of [13]-[72] of the DetailedDescription.

In some embodiments, the kits contain all of the components necessaryand/or sufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results. One skilled in the art willreadily recognize that the disclosed agents can be readily incorporatedinto one of the established kit formats which are well known in the art.

In some embodiments, the kits include a dosage amount (e.g., as used fortherapy or diagnosis) of at least one αPANTIFOL compositions (e.g., aαPANTIFOL liposome), or pharmaceutical formulation thereof, as disclosedherein. Kits may further comprise suitable packaging and/or instructionsfor use of the composition. Kits may also comprise a means for thedelivery for the composition, or pharmaceutical formulation thereof,such as a syringe for injection or other device as described herein andknown to those of skill in the art. One of skill in the art will readilyrecognize that the disclosed αPANTIFOL compositions can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits that comprise a αPANTIFOL compositions as wellas at least one additional therapeutic agent. In certain embodiments,the second (or more) therapeutic agent is an anti-metabolite. In certainembodiments, the second (or more) therapeutic agent is achemotherapeutic agent.

The following examples are intended to illustrate but not to limit thedisclosure in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may, be used without departing from the scope of the presentdisclosure.

FIGS. 1B-IN show chemical formulas of exemplary alpha polyglutamatesencompassed by the disclosure.

EXAMPLES Example 1: Liposomal Alpha Polyglutamated Antifolate(Pemetrexed) Compositions Methods Production of Alpha HexaglutamatedPemetrexed Liposomes

Briefly, L alpha hexaglutamated pemetrexed (aG6) and D alphahexaglutamated pemetrexed (aDG6) were encapsulated in liposomes by thefollowing procedure. First, the lipid components of the liposomemembrane were weighed out and combined as a concentrated solution inethanol at a temperature of around 65° C. In this example, the lipidsused were hydrogenated soy phosphatidylcholine, cholesterol, andDSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]). The molar ratio ofHSPC:Cholesterol:PEG-DSPE was approximately 3:2:0.15. Next, the aG6 oraDG6 was dissolved in 5% dextrose at a concentration of 150 mg/ml with apH of 6.5-6.9. The drug solution was heated up to 65° C. The ethanoliclipid solution was injected into the aG6 or aDG6 solution using asmall-bore needle. During this step the drug solution was well stirredusing a magnetic stirrer. The mixing was performed at an elevatedtemperature (63° C.-72° C.) to ensure that the lipids were in the liquidcrystalline state (as opposed to the gel state that they attain attemperatures below the lipid transition temperature Tm=51° C.-54° C.).As a result, the lipids were hydrated and form multiple bilayer(multilamellar) vesicles (MLV) containing aG6 or aDG6 in the aqueouscore.

Downsizing of MLV's Using Filter Extrusion

The MLVs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using three passes throughstacked (track-etched polycarbonate) membranes. The first pass wasperformed through stacked membranes consisting of two layers with a poresize of 200 nm. The remaining two passes were through the stackedmembranes consisting of three layers with a pore size of 100 nm. Duringextrusion, the temperature was maintained above the Tm to ensureplasticity of the lipid membranes. As a result of the extrusion, largeand heterogeneous in size and lamellarity MLVs turned into small,homogenous (90-125 nm) unilamellar vesicles (ULV) that sequestered thedrug in their interior. A Malvern Zetasizer Nano ZS instrument(Southborough, Mass.) with back scattering detector (90°) was used formeasuring the hydrodynamic size (diameter) at 25° C. in a quartz microcuvette. The samples were diluted 50-fold in formulation matrix beforeanalysis. Purification of liposomes

After the ULV's containing aG6 or aDG6 pemetrexed had been produced, theextra-liposomal gG6 was removed using columns for small volume ortangential flow diafiltration against a suitable buffer for largevolume. Although any buffer solution can be used, in this example thebuffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22 micron filter. The typical characteristics of liposomal derivativesare shown in the table below.

Starting Encapsulation Final Drug/Lipid Zeta con. efficiency con. RatioDiameter PDI potential Lps 1 mg/ml 4.75% 0.031 mg/ml 25-30 g/mol 122.8nm 0.021 −1.14 mV aDG6 Lps 1 mg/ml 5.90% 0.039 mg/ml 25-30 g/mol 100.2nm 0.018 −1.90 mV aG6 LpS 150 mg/ml  36% 8.0 mg/ml 230-260 g/mol 104 nm0.04 −2.73 mV aG6

Dose Response Study of Alpha HGP (Hexaglutamated Pemetrexed) andLiposomes

A dose response study was performed using liposomes containinghexaglutamated pemetrexed that were prepared essential as describedabove.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermines the number of viable cells in culture based on quantifyingATP that is present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curve were generated and fit using GraphPad Prism and IC50of each test article were calculated. A lower the IC50 is, the morepotent the test article is in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and 10 ng/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×10⁴cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results

In a set of dose response experiments, 6 cell lines representingdifferent types of cancers, namely HT-29 (colon cancer), H2342 (NSCLC,adenocarcinoma subtype), H292 (NSCLC, adenocarcinoma subtype), SW620(CRC), H1806 (triple negative breast cancer) and OAW28 (ovarian cancer),were studied (FIG. 2). Treatment consisted of exposure for 48 hoursusing 2 different encapsulated derivatives of liposomal alpha pemetrexedhexaglutamate, namely liposomal alpha L hexaglutamate (liposomal aG6)and its mirror image, liposomal alpha D hexaglutamate (liposomal aDG6)also referred to as its corresponding enantiomer.

The relative potency of the above mentioned derivatives as compared topemetrexed, following exposure over 48 hours, is represented in FIG. 2.The relative potency of treatment using the various derivatives, asshown in this figure was calculated by dividing the IC50 of pemetrexedby the IC50 of the liposomal alpha pemetrexed hexaglutamate for eachcell line. As shown in this figure, in all cell lines, the potency ofliposomal alpha pemetrexed hexaglutamate well exceeded that ofpemetrexed. By way of example, consider the NSCLC cell line H292. Asshown in the figure, the potency of liposomal alpha pemetrexedhexaglutamate was ≥50-fold that of pemetrexed. This suggests that a 2%or lower dose of the liposomal alpha pemetrexed hexaglutamate could havethe same treatment effect as a 100% dose of pemetrexed.

In some embodiments, increased uptake of payload is achieved bytargeting the liposomal delivery vehicle using antibody such as FolateReceptor Alpha. By way of example in the next two experiments LiposomalL Gamma G6/Lps Hexa gG6 was encapsulated using the methods describedabove. Subsequently, pemetrexed, liposomal gamma pemetrexedhexaglutamate derivatives (Liposomal L gamma G6/Lps Hexa gG6) and FolateReceptor Alpha Targeted Liposomal L Gamma G6 (Liposomal gG6-FR1Ab), Free(unencapsulated) L gamma G6 were tested for cytotoxic activity onrepresentative cell lines in non small cell lung cancer cells(NCI-H2342) and colorectal cancer cells (HT-29) as shown in FIG. 3 andFIG. 4 respectively. These data show that both liposomal L gammapemetrexed hexaglutamate and Folate Receptor Alpha Targeting liposomal Lgamma pemetrexed hexaglutamate are more potent than pemetrexed in bothcell lines. In general Folate Receptor Alpha Antibody targetingliposomes show the highest potency. By contrast free L gamma G6 has thelowest potency due to its inability to traffic across cell membraneseffectively.

Cancer cell viability studies comparing the liposomal alpha pemetrexedhexaglutamate derivatives (liposomal L alphaG6/Lps Hexa aG6 andliposomal D alphaG6/Lps Hexa aDG6) and pemetrexed for cytotoxic activityon representative cell lines in breast, lung and ovarian cancer areshown in FIGS. 5-7. These data show that both liposomal alpha Lpemetrexed hexaglutamate and liposomal alpha D pemetrexed hexaglutamateare more potent than pemetrexed. Further, as an indicator of efficacy,the results of the experiments on the same cell lines depicted atvarious dose levels ranging from 16 to 128 nM in FIGS. 8-10. As shown inthese figures, at each of these dose ranges, liposomal alpha Lpemetrexed hexaglutamate and liposomal alpha D pemetrexed hexaglutamateare superior to pemetrexed in terms of inhibiting cancer cells for thelung and breast cancer cell lines. In the ovarian cancer cell line,pemetrexed at the dose of 128 nM, appears to be equally effective asliposomal alpha pemetrexed hexaglutamate, whereas the liposomal alphapemetrexed hexaglutamate at the dose of 32 nM and 64 nM has a bettertreatment effect than pemetrexed; at 16 nM the treatment effect is lowerand similar in magnitude for liposomal alpha pemetrexed hexaglutamateand pemetrexed.

As shown in FIGS. 21A-F, each of liposomal pemetrexed alpha-Ltetraglutamate (Liposomal aG4), liposomal pemetrexed alpha-Lhexaglutamate (Liposomal aG6), liposomal pemetrexed alpha-Loctaglutamate (Liposomal aG8), and liposomal pemetrexed alpha-Ltridecaglutamate (Liposomal aG13), demonstrate a dose dependent effectin treating H2342 (NSCLC, adenocarcinoma subtype) cells, H292 (NSCLC,adenocarcinoma subtype) cells, HT-29 (colon cancer) cells, HCC1806(triple negative breast cancer) cells, MCF7 (ER+ breast cancer) cells,and OAW28 (ovarian cancer) cells, respectively, over 48 hours.

The major toxicities seen in patients treated with pemetrexed is bonemarrow suppression which manifests as a decrease in blood countsincluding neutrophil counts (a type of white blood cells). There is alsosome adverse effect on the lining of the mouth and gut that manifests asdiarrhea and mucositis, as well as an adverse effect on the liver insome instances. To assess the above-mentioned toxicities, treatment ofthe liposomal alpha pemetrexed hexaglutamate derivatives (L and D) andpemetrexed was measured at 48 hours on CD34+ cells that weredifferentiated into neutrophils, CCD841 colon epithelium cells and AML12liver cells. As shown in FIG. 11, liposomal alpha pemetrexedhexaglutamate is significantly less toxic to differentiating humanneutrophils in contrast to pemetrexed. This is also supported byneutrophil counts that are better preserved following treatment with theliposomal alpha L pemetrexed hexaglutamate or liposomal alpha Dpemetrexed hexaglutamate compared to pemetrexed, at dose ranges from 16nM to 128 nM (FIG. 12). Strikingly, there does not appear to be anytoxicity to the liver cells following treatment with liposomal L alphapemetrexed hexaglutamate or liposomal alpha D pemetrexed hexaglutamateat the dose levels studied (FIG. 13). In contrast, pemetrexed at alldoses studied is leading to a reduction in the liver cell counts ofapproximately 40%. And finally, the same trend is seen followingtreatment of epithelial colon cells (FIG. 14). As shown in this figure,pemetrexed at all doses studied is leading to approximately a ≥50%decrease in the number of cells compared to approximately a 20% or lessdecrease after treatment with liposomal alpha L pemetrexed hexaglutamateand liposomal alpha D pemetrexed hexaglutamate.

Example 2: Polyglutamated Pemetrexed-Cisplatin Complexes (PGPD) Methods:

Folate Analogs also known as antifolates have been an importantanticancer treatment for the last 70 years. Used in this setting thisclass of anti-cancer drugs interferes with various enzymes in theimportant folate metabolic pathway. This can result in impairedpyrimidine and purine (DNA and RNA) synthesis, impaired amino acidglycine and serine metabolism, impaired redox response and impairedmethylation processes within the cell.

In clinical practice, Antifolates such as pemetrexed are often used incombination with platinum agents such as cisplatin and carboplatin. Thecombinations result in enhanced efficacy. In this context, we set out tocoencapsulated the polyglutamates with platinum agents in a specificratio to facilitate controlled delivery of a predetermined ratio of thetwo anticancer drugs namely a polyglutamated Antifolate and a platinumanalog. We surprisingly discovered that long forms of polyglutamateantifolate (e.g., pentaglutamated Antifolate) forms a complex withcisplatin that is stable at high pH, and that this complex disassociatesinto polyglutamate and cisplatin at low pH. Low pH is believed to beoccur in many tumor cells and the tumor cell environment, particularlyin hypoxic settings. Application of this discovery provides the abilityto facilitate the delivery of combinations of alpha polyglutamatedAntifolates and therapeutic agents such as cisplatin to target cellssuch as tumor cells and to release the drugs from the complex inphysiologically relevant low pH conditions.

Production of Polyglutamated Pemetrexed-DDAP (Cisplatin) Complexes(PGPD)

To produce a polyglutamated Antifolate complex (Polyglutamatedpemetrexed-cisplatin DDAP Complex), alpha hexaglutamate (aG6) andDiammine dicarboxylic acid platinum (DDAP) was used. The process ofcomplexation was dependent on the presence of chlorinated platinumcompound and pH conditions. The complexation was achieved by anucleophilic attack on one or two carboxyl groups of glutamate by theplatinate derivative. Briefly the complex was formed by the followingprocedure. First, the active compound DDAP was weighed and dissolved in5% dextrose. After the DDAP dissolution step, aG6 was weighed out andadded to the DDAP-Captisol® (solution and allowed to stir for 1 hour at45-55° C. The pH of the solution was adjusted to 6.5-7.0 using 1N NaOHand the solution was stirred for 1-2 hour. The formation of complex wasconfirmed visually. However when the pH is adjusted to acidic pH of 3-5,the color reverted back to its original, indicating the decomplexatoinof the polyglutamated pemetrexed and cisplatin. FIG. 15 depicts aschematic providing possible scenarios explaining the observed pHdependent complex formation between the polyglutamated pemetrexed andcisplatin.

Complex formation was confirmed using HPLC which showed two distinctpeaks that merge into 1 large peak at high pH of 6.5 to 7.5 and thenreappear at low pH of 3-5. Repeating the experiment without Captisolshowed that complex formation was independent of Captisol®.

Production of Pentaglutamated Pemetrexed-DDAP Complex (PGPD) Liposomes

Briefly PGPD was encapsulated in liposomes by the following procedure.First, the lipid components of the liposome membrane was weighed out andcombined as a concentrated solution in ethanol at a temperature ofaround 65° C. In this example, the lipids used were hydrogenated soyphosphatidylcholine, cholesterol, and DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]). The molar ratio of HSPC:Cholesterol:PEG-DSPE wasapproximately 3:2:0.15. Next, PGPD was prepared as described above. ThePGPD drug solution was heated up to 65° C. The ethanolic lipid solutionwas injected into the PGPD solution using a small-bore needle. Duringthis step the drug solution was well stirred using a magnetic stirrer.The mixing was performed at an elevated temperature (63° C.-72° C.) toensure that the lipids were in the liquid crystalline state (as opposedto the gel state that they attain at temperatures below the lipidtransition temperature Tm=51° C.-54° C.). As a result, the lipids werehydrated and formed multiple bilayer (multilamellar) vesicles (MLV)containing PGPD in the aqueous core.

Downsizing of MLV's Using Filter Extrusion:

The MLVs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using two passes throughstacked (track-etched polycarbonate) membranes. The stacked membraneshave two layers with a pore size of 200 nm and six layers with a poresize of 100 nm. During extrusion, the temperature was maintained abovethe Tm to ensure plasticity of the lipid membranes. Because of theextrusion, large and heterogeneous in size and lamellarity MLVs turninto small, homogenous (100-120 nm) unilamellar vesicles (ULV) thatsequester the drug in their interior. A Malvern Zetasizer Nano ZSinstrument (Southborough, Mass.) with back scattering detector (90°) wasused for measuring the hydrodynamic size (diameter) at 25° C. in aquartz micro cuvette. The samples were diluted 50-fold in formulationmatrix before analysis.

Purification of Liposomes:

After the ULV's containing PGPD had been produced, the extra-liposomalPGPD was removed using columns for small volume or tangential flowdiafiltration against a suitable buffer for large volume. Although manydifferent buffers known in the art could have been used, in this examplethe buffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22-micron filter. The liposomes prepared according to the aboveprocedures were determined to have a diameter of 116.6 nm, a PDI of0.083, and a zeta potentials of −2.05 mV.

Example 3: Targeted Liposome Polyglutamated Pemetrexed Cell DeliveryMethods: Production of Targeted Gamma Hexaglutamated Pemetrexed (HGP)Liposomes

Gamma HGP (gG6) was encapsulated in liposomes and the liposomes weredownsized and purified according to procedures essentially as set forthabove in Example 1.

Antibody Conjugation:

Activated liposomes were prepared by adding DSPE-PEG-maleimide to thelipid composition. The liposomes contain four different lipids:hydrogenated soy phosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody thiolation was accomplished through use of Traut's reagent(2-iminothiolane) to attach a sulfhydryl group onto primary amines.Antibody was suspended in PBS at a concentration of 0.9-1.6 mg/ml.Traut's reagent (14 mM) was added to antibody solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody was added toactivated liposomes at a ratio of 60 g/mol phosphate lipids, and thereaction mixture was incubated for one hour at room temperature andover-night at 4 uL-cysteine was used to terminate the reaction andunconjugated antibody was removed through dialysis.

Exemplary direct and post insertion antibody-liposome conjugationmethods are provided below.

Exemplary Antibody Conjugation Method 1: Direct Conjugation

Antibody or its fragments, such as Fab or scFv, can be conjugateddirectly onto thiol-reactive liposome. Thiol-reactive liposomes areprepared by adding DSPE-PEG-maleimide to the lipid composition. Theliposomes contain four different lipids: hydrogenated soyphosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody (or its fragments, such as Fab or scFv) thiolation isaccomplished through use of Traut's reagent (2-iminothiolane) to attacha sulfhydryl group onto primary amines. Antibody (or its fragment) issuspended in PBS at a concentration of 0.9-1.6 mg/ml. Traut's reagent(14 mM) is added to antibody (or its fragment) solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody (or its fragment) isadded to thiol-reacitve liposome at a ratio of 60 g/mol phosphatelipids, and the reaction mixture is incubated for one hour at roomtemperature and over-night at 4° C. L-cysteine is used to terminate thereaction and unconjugated antibody (or its fragment) is removed throughdialysis.

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated directly onto theliposome by incubating a reduced antibody (or its fragment) withthiol-reactive liposome. Antibody (or its fragment) with a cysteine tailis dissolved and reduced by a 10-20 mM reducing reagent (such as2-mercaptoethylamine, cysteine, or dithioerythritol) at pH<7. The excessreducing reagent is removed thoroughly by size exclusion chromatographyor dialysis. The purified and reduced antibody (or its fragment) can bedirectly conjugated to the thiol-reactive liposome.

Exemplary Antibody Conjugation Method 2: Post Insertion:

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated and incorporatedinto the liposome through a “post insertion” method. Micelles ofthiol-reactive lipopolymer (such as DSPE-PEG-maleimide) is prepared bydissolving in an aqueous solution at 10 mg/ml. Antibody (or itsfragment) with a cysteine tail is dissolved and reduced by a 10-20 mMreducing reagent (such as 2-mercaptoethylamine, cysteine, ordithioerythritol) at pH<7. The excess reducing reagent is removedthoroughly by size exclusion chromatography or dialysis. The purifiedand reduced antibody (or its fragment) is then incubated with themicelles of thiol-reactive lipopolymers at a molar ratio of 1:4. At theend of the reaction, the excess maleimide groups are quenched by a smallamount of cysteine (1 mM) or mercaptoethanol. Unconjugated antibody (orits fragment) is removed by size exclusion chromatography. Purifiedconjugated micelles is then incubated with liposome at 37° C. orelevated temperature.

Physical Characteristics of the Nanoparticles

Starting Encapsulation Final Drug/Lipid Zeta con. efficiency con. RatioDiameter PDI potential Lps 20 mg/ml 10.60% 1.39 mg/ml 35-50 g/mM 114.9nm 0.035 −1.76 mV gG6 lipidsDose response study of HGP (pentaglutamated pemetrexed) and liposomes.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermines the number of viable cells in culture based on quantifyingATP that was present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curves were generated and fit using GraphPad Prism andIC50 of each test article were calculated. A lower the IC50 is, the morepotent the test article was in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and 10 ng/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×10⁴cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results:

The dose response relationship of free pemetrexed gamma hexaglutamate(gG6), (non-targeted) liposomal gamma hexaglutamate (liposomal gG6),pemetrexed and folate receptor alpha targeting antibody (FR1Ab)liposomal pemetrexed gamma hexaglutamate (liposomal gG6-FR1Ab), in theNCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype isshown in FIG. 3. The output is percentage of viable cells after 48 hoursof treatment as measured by luciferase luminescence. As shown in thisFIG. 3, the free pemetrexed gG6 appears to be the least potent asmeasured by IC50. Both the liposomal pemetrexed gG6 and the liposomalpemetrexed gG6-FR1Ab are 7-fold and 40-fold more potent, respectively,than free pemetrexed.

Similar data is shown for the HT-29 colon cancer cell line in FIG. 4that depict cell viability expressed as a percentage. As shown in thisfigure, free pemetrexed gG6 appears to be the least potent. In thisinstance, the liposomal pemetrexed gG6 is twice as potent as pemetrexedand the liposomal pemetrexed gG6-FR1Ab is 5-fold more potent than freepemetrexed.

Example 4: In Vivo Studies Methods Safety Studies in Mice

Because some of the major toxicities associated with a pemetrexed basedtreatment are hematologic and hepatic, it is important to evaluate theeffect of Liposomal alpha G6 (Lp-aG6) in an in-vivo (murine) model andcompare the changes in hematologic and the liver serum chemistry panelfollowing treatment. To obtain this data an initial dose ranging studywas conducted using healthy female BALB/c mice (6-8 weeks old) whichwere purchased from The Jackson Laboratory (Bar Harbor, Me.). Prior tothe study, animals were weighed, randomized by weight, observed forclinical abnormalities, and distributed into groups (5 mice per group).Doses from 10 mg/kg up to 200 mg/kg were investigated to identify atolerable dose in mice. Treatments were administrated intravenously oncea week for four weeks. Body weight and detailed clinical observationwere recorded daily. At the end of study, Day 28, mice were euthanized,and blood and tissue were harvested from untreated Control mice and forthe mice treated with Liposomal aG6 40 mg/kg and Liposomal aG6 80 mg/kg.Whole blood was collected into K2-EDTA anticoagulant tubes forcomprehensive complete blood count (CBC) and serum was isolated forcomprehensive chemistry and was sent to IDEXX (Westbrook, Me.) on theday of collection.

Results

In general, treatment with once weekly liposomal aG6 at two dose levelsof 40 mg/kg and 80 mg/kg for 4 weeks was well tolerated and there wereno major differences in weight compared to untreated controls. To assesssome of the effects on hematologic parameters, white blood cell (WBC)counts, neutrophil counts as well as platelet counts were measured aftertreatment with liposomal aG6 at two dose levels of 40 mg/kg and 80 mg/kgboth given once weekly for 4 weeks. As can be seen in FIG. 16, therewere no appreciable decreases in mean neutrophil, mean white blood celland mean platelet counts, after four weeks of treatment with LiposomalaG6 in treated animals compared to untreated control animals. Hemoglobinand reticulocyte indices were measured to assess the impact on red bloodcell. As shown in FIG. 17, there was a minimal decrease in meanhemoglobin concentrations at the higher dose level. In parallel there isa slight increase in mean reticulocytosis indices which suggests a bonemarrow's response to treatment by increasing red blood cell production.Altogether this effect seems minor as the mice hemoglobin levels aremaintained after 4 weeks of treatment. Taken together these data suggestthat at these dose levels, 40 mg/kg and 80 mg/kg once-weekly, there islittle impact on the bone marrow and related hematologic indices.

Another concern with pemetrexed is hepatic toxicity that has beenobserved in some patients treated with pemetrexed based therapy. Toassess hepatic well-being in mice serum chemistries including serumaspartate transaminase (AST) and serum alanine transaminase (ALT) alongwith serum albumin were measured. As shown in FIG. 18, there were noappreciable increases in liver transaminases mean AST and mean ALTlevels at 4 weeks following treatment with Liposomal aG6 at the two doselevels of 40 mg/kg and 80 mg/kg both given once weekly for 4 weeks whencompared to untreated controls. There was no change in mean albuminlevels either. Taken together these data suggest a favorable safetyprofile for Liposomal aG6.

Preliminary Pilot Efficacy Study in Mice Xenografts

To assess whether there was any tumor control following treatment withliposomal alpha pemetrexed G6 (Lp-aG6) the pilot study was conducted. Inthis study immunodeficient female Nude mice (Nu/J; 6-8 weeks old) werepurchased from The Jackson Laboratory (Bar Harbor, Me.). NCI-H292(Non-Small Cell Lung Cancer) cells were cultured in RPMI mediasupplemented with 10% Fetal Bovine Serum in a 37° C., 5% CO₂ incubator.1×10⁶ cells were inoculated subcutaneously into the dorsal hind flank ofeach mouse. Tumor volume and body weight were monitored twice everyweek. Tumor-bearing mice were randomized by tumor volume on Day 0 anddistributed into groups (5 mice per group): Control, Pemetrexed, andLiposomal aG6. Pemetrexed was given intravenously at 167 mg/kg onceevery three weeks. This murine dose of 167 mg/kg every three weeks isequivalent to the FDA/EMA approved human dose and schedule of 500 mg/m²every three weeks. Liposomal aG6 was dosed intravenously at 80 mg/kgonce a week for four weeks. Tumor size was measured with a caliper andtumor burden is calculated using the following equations: tumorvolume=0.5×(tumor length)×(tumor width)²; Relative tumor volume=(tumorvolume/tumor volume on Day 0)×100%. This study is still ongoing butpreliminary data are shown in FIG. 19. In this figure, relative tumorvolume is displayed following treatment with Liposomal aG6 andpemetrexed. As can be seen from these preliminary data, liposomal aG6provides better tumor control when compared to pemetrexed.

In a parallel study, the survival of mice (10) dosed intravenously with90 mg/kg Liposomal aG6 once a week for four weeks (90 mg/kgintravenously one a week for 6 weeks), mice (10) dosed with pemetrexed(167 mg/kg intravenously every three weeks for 6 weeks) was studied inthe NSCLC (H292) xenograft model. The results of this study arepresented in FIG. 20 and the table provide below. As indicated in thetable presented below, the median survival following treatment withLiposomal aG6 was longer than that following treatment with pemetrexed.Also, 50% of the mice treated with Liposomal aG6 were alive at 60 dayspost dosing, whereas none of mice treated with pemetrexed survived to 60days.

Median Survival (days) 60 Day Survival Control 28.5 0% Pemetrexed 39.00% Liposomal aG6 58.5 50% 

Further Embodiments

In a non-limiting embodiment, of this disclosure, there is provided acomposition comprising alpha polyglutamated Antifolate.

In the composition of the immediately preceding paragraph, thecomposition may comprise pentaglutamated or hexaglutamated Antifolate.

In the composition of any of the preceding two paragraphs, thecomposition may comprise alpha polyglutamated Antifolate which mayinclude pentaglutamated or hexaglutamated Antifolate.

A non-limiting example liposomal alpha polyglutamated Antifolate(L-αPANTIFOL) composition may comprise a composition of any of thepreceding three paragraphs and the liposome may be optionally pegylated(PL-αPANTIFOL).

In the L-αPANTIFOL or PL-αPANTIFOL composition of the immediatelypreceding paragraph, the alpha polyglutamated Antifolate may includepentaglutamated or hexaglutamated Antifolate.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingtwo paragraphs, the liposome may be anionic or neutral.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingthree paragraphs, a targeting moiety may be attached to one or both of aPEG and the exterior of the liposome, and the targeting moiety may havea specific affinity for a surface antigen on a target cell of interest(TL-αPANTIFOL or TPL-αPANTIFOL).

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingfour paragraphs, a targeting moiety may be attached to one or both of aPEG and the exterior of the liposome and may be a polypeptide.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingfive paragraphs, a targeting moiety may be attached to one or both a PEGand the exterior of the liposome and may be an antibody or a fragment ofan antibody.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingsix paragraphs, one or more of an immunostimulatory agent, a detectablemarker and a maleimide may be disposed on at least one of a PEG and theexterior of the liposome.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingseven paragraphs, a polypeptide may bind an antigen with an equilibriumdissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to 10×10⁻⁶ asdetermined using BIACORE® analysis.

In the L-αPANTIFOL or PL-αPANTIFOL composition of any of the precedingeight paragraphs, a polypeptide may specifically bind one or more folatereceptors selected from: folate receptor alpha (FR-α), folate receptorbeta (FR-β), and folate receptor delta (FR-δ).

A non-limiting exemplary method of killing a hyperproliferative cellthat includes contacting a hyperproliferative cell with a liposomalalpha polyglutamated Antifolate composition of any of the preceding nineparagraphs.

In the method of the immediately preceding paragraph, thehyperproliferative cell is a cancer cell.

A non-limiting example method for treating cancer comprisesadministering an effective amount of the alpha polyglutamated Antifolatecomposition of any of preceding paragraphs from preceding paragrapheleven to preceding paragraph three, to a subject having or at risk ofhaving cancer.

In the method of the immediately preceding paragraph, the cancer may beone or more selected from: lung cancer, pancreatic, breast cancer,ovarian cancer, lung cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colon cancer, esophagealcancer, cervical cancer, kidney cancer, biliary duct cancer, gallbladdercancer, and a hematologic malignancy. In some embodiments, the cancer isselected from: breast cancer, advanced head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, chorioadenoma, nonleukemic meningeal cancer,soft tissue sarcoma (desmoid tumors, aggressive fibromatosis), bladdercancer, and central nervous system (CNS) lymphoma.

A non-limiting example maintenance therapy for subjects that areundergoing or have undergone cancer therapy includes administering aneffective amount of the alpha polyglutamated Antifolate composition ofany of preceding paragraphs from preceding paragraph thirteen topreceding paragraph five, to a subject that is undergoing or hasundergone cancer therapy.

A non-limiting example pharmaceutical composition may include any alphapolyglutamated Antifolate composition of any of Section IV.

A non-limiting example method for treating a disorder of the immunesystem may include administering an effective amount of the of the alphapolyglutamated Antifolate composition of any of preceding paragraphsfrom preceding paragraph fourteen to preceding paragraph six, to asubject having or at risk of having a disorder of the immune system.

A non-limiting example method for treating an infectious may includecomprises administering an effective amount of the of the alphapolyglutamated Antifolate composition of any of preceding paragraphsfrom preceding paragraph fifteen to preceding paragraph seven, to asubject having or at risk of having an infectious disease.

A non-limiting example method of delivering alpha polyglutamatedAntifolate to a tumor expressing a folate receptor on its surface mayinclude administering a polyglutamated Antifolate composition of any ofpreceding paragraphs from preceding paragraph sixteen to precedingparagraph eight, to a subject having the tumor in an amount to deliver atherapeutically effective dose of the alpha polyglutamated Antifolate tothe tumor.

A non-limiting example method of preparing a liposomal alphapolyglutamated Antifolate composition which includes alphapolyglutamated Antifolate composition of any of preceding paragraphsfrom preceding paragraph seventeen to preceding paragraph nine includesforming a mixture comprising: liposomal components; alpha polyglutamatedAntifolate in solution; homogenizing the mixture to form liposomes inthe solution; and processing the mixture to form liposomes containingthe polyglutamated Antifolate.

A non-limiting example pharmaceutical composition includes an alphapolyglutamated Antifolate composition of any of preceding paragraphsfrom preceding paragraph eighteen to preceding paragraph ten.

Although the disclosure has been described with reference to varioussome embodiments, it should be understood that various modifications canbe made without departing from the spirit of the disclosure.Accordingly, the scope of the disclosure should be determined withreference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. Throughout thisapplication, various publications are referenced by author name anddate, or by patent No. or Patent Publication No. The disclosure of thesepublications are hereby incorporated in their entireties by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein. However, the citation of a referenceherein should not be construed as an acknowledgement that such referenceis prior art to the present invention.

Various new chemical entities, methods and equipment for making thesechemical entities are set forth below in the appended claims.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments, of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The disclosure of each of U.S. Appl. No. 62/627,703, filed Feb. 7, 2018;U.S. Appl. No. 62/627,714, filed Feb. 7, 2018; U.S. Appl. No.62/627,716, filed Feb. 7, 2018; U.S. Appl. No. 62/627,731, filed Feb. 7,2018; U.S. Appl. No. 62/627,741, filed Feb. 7, 2018; U.S. Appl. No.62/630,629, filed Feb. 14, 2018; U.S. Appl. No. 62/630,634, filed Feb.14, 2018; U.S. Appl. No. 62/630,637, filed Feb. 14, 2018; U.S. Appl. No.62/630,671, filed Feb. 14, 2018; U.S. Appl. No. 62/630,713, filed Feb.14, 2018; U.S. Appl. No. 62/630,728, filed Feb. 14, 2018; U.S. Appl. No.62/630,744, filed Feb. 14, 2018; U.S. Appl. No. 62/630,820, filed Feb.14, 2018; U.S. Appl. No. 62/630,825, filed Feb. 14, 2018; U.S. Appl. No.62/636,294, filed Feb. 28, 2018; U.S. Appl. No. 62/662,374, filed Apr.25, 2018; U.S. Appl. No. 62/702,732, filed Jul. 24, 2018; U.S. Appl. No.62/702,561, filed Jul. 24, 2018; U.S. Appl. No. 62/764,943, filed Aug.17, 2018; and U.S. Appl. No. 62/764,955, filed Aug. 17, 2018; is hereinincorporated by reference in its entirety.

1. A liposomal composition comprising an alpha polyglutamated Antifolateencapsulated by a liposome, wherein at least one glutamyl group of thepolyglutamated antifolate has an alpha carboxyl group linkage. 2.(canceled)
 3. The liposomal composition of claim 1, wherein theAntifolate is selected from: PMX, MTX, RTX, and LMX, or a stereoisomerthereof.
 4. The liposomal composition of claim 1, wherein the Antifolateis selected from: piritrexim, pralatrexate, AG2034, GW1843, andLY309887, or a stereoisomer thereof; LV (etoposide), L-leucovorin(L-5-formyltetrahydrofolate); 5-CH3-THF, 5-methyltetrahydrofolate; FA,folic acid; PteGlu, pteroyl glutamate (FA); MTX, methotrexate; 2-dMTX,2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin;2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM,10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-Ndelta-(hemiphthaloyl)-L-ornithine; DDATHF (lometrexol),5,10-dideaza-5,6,7,8-tetrahydrofolic acid; 5-d(i)H4PteGlu,5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu,N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, Nalpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, Nalpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dPteOrn, Nalpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphobutanoicacid; 5-dH4PteOro, Nalpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717,N10-propargyl-5,8-dideazafolic acid; ICI-198,583,2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid;4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583,4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-amino-suberate-ICI-198,583; 7-CH3-ICI-198,583,7-methyl-ICI-198,583; ZD1694,N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamicacid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89,(S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaricacid; LY231514,N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamicacid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ;2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaaisofolicacid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid;N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337,3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio) quanazoline; and2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or astereoisomer thereof; and methotrexate, raltitrexed, plevitrexed,pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), acyclopenta[g]quinazoline with a dipeptide ligand, CB3717, CB300945, or astereoisomer thereof, such as 6-R,S-BGC 945 (ONX-0801), CB300638, andBW1843U89.
 5. (canceled)
 6. The liposomal composition of claim 1,wherein, (a) each of the glutamyl groups of the polyglutamatedAntifolate other than the glutamyl group of the Antifolate has an alphacarboxyl group linkage; (b) two or more glutamyl groups of thepolyglutamated Antifolate have a gamma carboxyl group linkage; (c) eachof the glutamyl groups other than the C-terminal glutamyl group orgroups and the glutamyl group of the Antifolate has an alpha carboxylgroup linkage; or (d) each of the glutamyl groups other than theC-terminal glutamyl group or groups has an alpha carboxyl group linkage.7.-8. (canceled)
 9. The liposomal composition of claim 1, wherein thealpha polyglutamated Antifolate comprises 1-10 glutamyl groups having analpha carboxyl group linkage.
 10. The liposomal composition of claim 1,wherein: (a) at least 2 of the glutamyl groups of the alphapolyglutamated Antifolate are in the L-form, (b) each of the glutamylgroups of the alpha polyglutamated Antifolate is in the L-form, (c) atleast 1 of the glutamyl groups of the alpha polyglutamated Antifolate isin the D-form, (d) each of the glutamyl groups of the alphapolyglutamated Antifolate other than the glutamyl group of theAntifolate is in the D-form, or (e) at least 2 of the glutamyl groups ofthe alpha polyglutamated Antifolate are in the L-form and at least 1 ofthe glutamyl groups is in the D-form.
 17. The liposomal composition ofclaim 1, wherein the liposome comprises an alpha polyglutamatedAntifolate containing 4, 5, 6, 2-10, 4-6, or more than 5, glutamylgroups.
 18. The liposomal composition of claim 1, wherein the liposomecomprises an alpha tetraglutamated Antifolate.
 19. The liposomalcomposition of claim 1, wherein the liposome comprises an alphapentaglutamated Antifolate.
 20. The liposomal composition of claim 1,wherein the liposome comprises an alpha hexaglutamated Antifolate.21.-24. (canceled)
 25. The liposomal composition of claim 1, wherein theliposome is pegylated.
 26. The liposomal composition of claim 1, whereinthe liposome is not pegylated.
 27. (canceled)
 28. The liposomalcomposition of claim 1, wherein the polyglutamate is linear or branched.29. (canceled)
 30. The liposomal composition of claim 1, wherein theliposome has a diameter in the range of 20 nm to 500 nm, 20 nm to 200nm, or 80 nm to 120 nm.
 31. (canceled)
 32. The liposomal composition ofclaim 1, wherein the liposome is formed from liposomal componentscomprising: at least one of an anionic lipid and a neutral lipid; atleast one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC;HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide; or atleast one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC;DSPE-PEG-maleimide; cholesterol; and HSPC. 33.-35. (canceled)
 36. Theliposomal composition of claim 1, wherein one or more liposomalcomponents further comprises at least one steric stabilizer selectedfrom: polyethylene glycol (PEG); poly-L-lysine (PLL);monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP);poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L amino-acid-based polymer; oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol.
 37. (canceled)
 38. The liposomalcomposition according to claim 36, wherein the steric stabilizer is PEGand the PEG has a number average molecular weight (Mn) of 200 to 5000daltons.
 39. The liposomal composition of claim 1, wherein the liposomeis anionic or neutral.
 40. The liposomal composition of claim 39,wherein the liposome has a zeta potential that is less than or equal tozero, between 0 to −150 mV, or between −30 to −50 mV. 41.-42. (canceled)43. The liposomal composition of claim 1, wherein the liposome iscationic.
 44. The liposomal composition of claim 1, wherein the liposomehas an interior space comprising the alpha polyglutamated Antifolate andan aqueous pharmaceutically acceptable carrier comprising: a tonicityagent such as, dextrose, mannitol, glycerine, potassium chloride, sodiumchloride, at a concentration of greater than 1%; 1% to 50% trehalose; 1%to 50% dextrose; 5% dextrose suspended in an HEPES buffered solution; ora total concentration of sodium acetate and calcium acetate of between50 mM to 500 mM. 45.-49. (canceled)
 50. The liposomal composition ofclaim 44, wherein the pharmaceutically acceptable carrier comprises abuffer such as HEPES Buffered Saline (HBS) or similar, at aconcentration of between 1 to 200 mM and a pH of between 2 to
 8. 51.(canceled)
 52. The liposomal composition of claim 44, wherein theinterior space of the liposome has a pH of 5-8 or a pH of 6-7, or anyrange therein between.
 53. The liposomal composition of claim 1, whereinthe liposome comprises less than 500,000, less than 200,000, or between10 to 100,000 molecules of the alpha polyglutamated Antifolate, or anyrange therein between.
 54. (canceled)
 55. The liposomal composition ofclaim 1, which further comprises a targeting moiety and wherein thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest.
 56. The liposomal composition of claim 55,wherein the targeting moiety is attached to one or both of a PEG and theexterior of the liposome, optionally wherein targeting moiety isattached to one or both of the PEG and the exterior of the liposome by acovalent bond.
 57. The liposomal composition of claim 55, wherein thetargeting moiety is a polypeptide, an antibody or an antigen bindingfragment of an antibody.
 58. (canceled)
 59. The liposomal composition ofclaim 55, wherein the targeting moiety binds the surface antigen with anequilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to10×10⁻⁶ as determined using BIACORE® analysis.
 60. The liposomalcomposition of claim 55, wherein the targeting moiety specifically bindsone or more folate receptors selected from: folate receptor alpha(FR-α), folate receptor beta (FR-β), and folate receptor delta (FR δ).61. The liposomal composition of claim 55, wherein the targeting moietycomprises one or more selected from: an antibody, a humanized antibody,an antigen binding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody, and a multimeric antibody.
 62. The liposomalcomposition of claim 25, wherein the pegylated liposome comprises from 1to 1000 or 30-200 targeting moieties.
 63. The liposomal composition ofclaim 25, further comprising one or more of an immunostimulatory agent,a detectable marker and a maleimide, wherein the immunostimulatoryagent, the detectable marker or the maleimide is attached to said PEG orthe exterior of the liposome.
 64. (canceled)
 65. The liposomalcomposition of claim 63, wherein the immunostimulatory agent is at leastone selected from: a fluorescein; a fluorescein isothiocyanate (FITC); aDNP; a beta glucan; a beta-1,3-glucan; a beta-1,6-glucan; a resolvin(e.g., a Resolvin D such as Dn-6DPA or Dn-3DPA, a Resolvin E, or a Tseries resolvin); and a Toll-like receptor (TLR) modulating agent suchas, an oxidized low-density lipoprotein (e.g. OXPAC, PGPC), and aneritoran lipid (e.g., E5564). 66.-68. (canceled)
 69. The liposomalcomposition of claim 1, which further comprises at least onecryoprotectant selected from mannitol; trehalose; sorbitol; and sucrose.70.-71. (canceled)
 72. The liposomal composition of claim 1, whichfurther comprises carboplatin and/or pembroluzumab.
 73. A pharmaceuticalcomposition comprising the liposomal composition of claim
 1. 74.-77.(canceled)
 78. A method for treating or preventing disease in a subjectneeding such treatment or prevention, the method comprisingadministering the liposomal composition of claim 1 to the subject. 79.(canceled)
 80. A method of killing a hyperproliferative cell thatcomprises contacting a hyperproliferative cell with the liposomalcomposition of claim
 1. 81. The method of claim 80, wherein thehyperproliferative cell is a cancer cell, a mammalian cell, and/or ahuman cell.
 82. (canceled)
 83. A method for treating cancer thatcomprises administering an effective amount of the liposomal compositionof claim 1 to a subject having or at risk of having cancer.
 84. Themethod of claim 83, wherein the cancer is selected from: anon-hematologic malignancy including such as for example, lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldyscrasias; breast cancer, head and neck cancer, lung cancer, stomachcancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma)choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, softtissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer,and central Nervous System (CNS) lymphoma; mesothelioma and non-smallcell lung carcinoma (NSCLC); or a sarcoma such as osteosarcoma. 85.-87.(canceled)
 88. A method for treating cancer that comprises administeringan effective amount of the liposomal composition of claim 60 to asubject having or at risk of having a cancer cell that expresses on itssurface a folate receptor bound by the targeting moiety.
 89. (canceled)90. A maintenance therapy comprising administering an effective amountof the liposomal composition of claim 1 to a subject that is undergoingor has undergone cancer therapy.
 91. (canceled)
 92. A method fortreating a disorder of the immune system that comprises administering aneffective amount of the liposomal composition of claim 1 to a subjecthaving or at risk of having a disorder of the immune system.
 93. Amethod for treating: (a) an infectious disease, cardiovascular disease,metabolic disease, or another disease, that comprises administering aneffective amount of the liposomal composition of claim 1 to a subjecthaving or at risk of having an infectious disease, cardiovasculardisease, or another disease, wherein the disease is a member selectedfrom: atherosclerosis, cardiovascular disease (CVD), coronary arterydisease, myocardial infarction, stroke, metabolic syndrome, agestational trophoblastic disease, and ectopic pregnancy; (b) anautoimmune disease that comprises administering an effective amount ofthe liposomal composition of claim 1 to a subject having or at risk ofhaving an autoimmune disease; (c) rheumatoid arthritis that comprisesadministering an effective amount of the liposomal composition of claim1 to a subject having or at risk of having rheumatoid arthritis; (d) aninflammatory condition that comprises administering an effective amountof the liposomal composition of claim 1 to a subject having or at riskof having inflammation, optionally wherein the inflammation is acute,chronic, and/or systemic inflammation; or (e) a skin condition thatcomprises administering an effective amount of the liposomal compositionof claim 1 to a subject having or at risk of having a skin condition,optionally wherein the skin condition is psoriasis.
 94. A method fortreating an infectious disease that comprises administering an effectiveamount of the liposomal composition of claim 1 to a subject having or atrisk of having an infectious disease.
 95. A method of delivering alphapolyglutamated Antifolate to a tumor expressing a folate receptor on itssurface, the method comprising: administering the liposomal compositionof claim 60 to a subject having the tumor in an amount to deliver atherapeutically effective dose of the alpha polyglutamated Antifolate tothe tumor.
 96. A method of preparing an alpha polyglutamated Antifolatecomposition comprising the liposomal composition of claim 1, the methodcomprising: forming a mixture comprising: liposomal components and alphapolyglutamated antifolate in solution; homogenizing the mixture to formliposomes in the solution; and processing the mixture to form liposomescontaining alpha polyglutamated Antifolate.
 97. A method of preparing analpha polyglutamated Antifolate composition comprising the liposomalcomposition of claim 1, the method comprising: forming a mixturecomprising: liposomal components and alpha polyglutamated Antifolate insolution; and processing the mixture to form liposomes containing alphapolyglutamated Antifolate, optionally wherein the processing the mixturecomprises homogenizing the mixture to form liposomes in the solution.98.-99. (canceled)
 100. A method of preparing the liposomal compositionof claim 60, comprising the steps of: forming a mixture comprising:liposomal components and alpha polyglutamated Antifolate in a solution;processing the mixture to form liposomes entrapping and/or encapsulatingalpha polyglutamated Antifolate; and providing a targeting moiety on asurface of the liposomes, the targeting moiety having specific affinityfor at least one of folate receptor alpha (FR-α), folate receptor beta(FR-β) and folate receptor delta (FR-δ), optionally wherein theprocessing step comprises homogenizing the mixture to form liposomes inthe solution or wherein the processing step includes one or more stepsof: thin film hydration, extrusion, in-line mixing, ethanol injectiontechnique, freezing-and-thawing technique, reverse-phase evaporation,dynamic high pressure microfluidization, microfluidic mixing, doubleemulsion, freeze-dried double emulsion, 3D printing, membrane contactormethod, and stirring or wherein said processing step includes one ormore steps of modifying the size of the liposomes by one or more ofsteps of extrusion, high-pressure microfluidization, and/or sonication.101.-104. (canceled)